U.S. patent application number 17/226153 was filed with the patent office on 2021-10-21 for compounds specific to coronavirus s protein and uses thereof.
The applicant listed for this patent is Adagio Therapeutics, Inc.. Invention is credited to Jonathan Belk, Laura Deveau, C. Garrett Rappazzo, Laura Walker, Anna Wec.
Application Number | 20210324048 17/226153 |
Document ID | / |
Family ID | 1000005597542 |
Filed Date | 2021-10-21 |
United States Patent
Application |
20210324048 |
Kind Code |
A1 |
Walker; Laura ; et
al. |
October 21, 2021 |
COMPOUNDS SPECIFIC TO CORONAVIRUS S PROTEIN AND USES THEREOF
Abstract
The present disclosure is directed to antibodies, and antigen
binding fragments thereof, having binding specificity for the S
protein of coronaviruses (CoV-S), such as the S protein of the SARS
coronavirus (SARS-CoV-S) and/or the S protein of the SARS
coronavirus 2 (SARS-CoV-2-S), including neutralizing antibodies and
antibodies that bind to and/or compete for binding to the same
linear or conformational epitope(s) on CoV-S. Further disclosed are
conjugates of anti-CoV-S antibodies, and binding fragments thereof,
conjugated to one or more functional or detectable moieties.
Methods of making said anti-CoV-S antibodies and antigen binding
fragments thereof are also contemplated. Other embodiments of the
disclosure include the use of anti-CoV-S antibodies, and binding
fragments thereof, for the diagnosis, assessment, and treatment of
diseases and disorders associated with coronaviruses, or the S
protein thereof, and conditions where neutralization or inhibition
of coronaviruses, or the S protein thereof, would be
therapeutically and/or prophylactically beneficial.
Inventors: |
Walker; Laura; (Lebanon,
NH) ; Deveau; Laura; (Lebanon, NH) ; Belk;
Jonathan; (Lebanon, NH) ; Wec; Anna; (Lebanon,
NH) ; Rappazzo; C. Garrett; (Lebanon, NH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Adagio Therapeutics, Inc. |
Waltham |
MA |
US |
|
|
Family ID: |
1000005597542 |
Appl. No.: |
17/226153 |
Filed: |
April 9, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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63163400 |
Mar 19, 2021 |
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63152054 |
Feb 22, 2021 |
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63150413 |
Feb 17, 2021 |
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63148754 |
Feb 12, 2021 |
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63147495 |
Feb 9, 2021 |
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63143456 |
Jan 29, 2021 |
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63138886 |
Jan 19, 2021 |
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63112122 |
Nov 10, 2020 |
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63046313 |
Jun 30, 2020 |
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63021589 |
May 7, 2020 |
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63008545 |
Apr 10, 2020 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/565 20130101;
C07K 2317/55 20130101; C07K 16/10 20130101; C07K 2317/76 20130101;
C07K 2317/622 20130101; C07K 2317/54 20130101; C07K 2317/92
20130101 |
International
Class: |
C07K 16/10 20060101
C07K016/10 |
Claims
1.-46. (canceled)
47. An isolated antibody, or antigen-binding fragment thereof,
which specifically binds to the spike protein of a coronavirus
("CoV-S"), wherein said antibody, or antigen-binding fragment
thereof, comprises a heavy chain variable region (VH) comprising a
VH CDR1 comprising SEQ ID NO:22304, a VH CDR2 comprising SEQ ID
NO:22306, and a VH CDR3 comprising SEQ ID NO:22308, and a light
chain variable region (VL) comprising a VL CDR1 comprising SEQ ID
NO:22314, a VL CDR2 comprising SEQ ID NO:22316, and a VL CDR3
comprising SEQ ID NO:22318.
48. The isolated antibody, or antigen-binding fragment thereof, of
claim 47, wherein the VH comprises SEQ ID NO:22302 and the VL
comprises SEQ ID NO:22312.
49. The isolated antibody, or antigen-binding fragment thereof, of
claim 48, wherein the VH consists of SEQ ID NO:22302 and the VL
consists of SEQ ID NO:22312.
50. The isolated antibody, or antigen-binding fragment thereof, of
claim 47, wherein the CoV-S is the spike protein of SARS-CoV
("SARS-CoV-S") or the spike protein of SARS-CoV-2
("SARS-CoV-2-S").
51. The isolated antibody, or antigen-binding fragment thereof, of
claim 47, wherein the antibody, or antigen-binding fragment
thereof, cross-reacts with SARS-CoV-S and SARS-CoV-2-S.
52. The isolated antibody, or antigen-binding fragment thereof, of
claim 51, wherein SARS-CoV-S comprises an amino acid sequence of
SEQ ID NO:1, and wherein SARS-CoV-2-S comprises an amino acid
sequence of SEQ ID NO:5.
53. The isolated antibody, or antigen-binding fragment thereof, of
claim 51, wherein the SARS-CoV-2-S is a B.1.1.7 variant, a B.1.351
variant, a B.1.1.28 variant, or a B. 1.429 variant of SEQ ID
NO.5.
54. The isolated antibody, or antigen-binding fragment thereof, of
claim 47, which neutralizes SARS-CoV and/or SARS-CoV-2.
55. The isolated antibody, or antigen-binding fragment thereof, of
claim 54, which neutralizes SARS-CoV and/or SARS-CoV-2 with an IC50
of about 100 nM or lower.
56. The isolated antibody, or antigen-binding fragment thereof, of
claim 47, which binds to SARS-CoV-S with a KD value of about 100 nM
or lower.
57. The isolated antibody, or antigen-binding fragment thereof, of
claim 47, wherein the antibody is human, humanized, primatized,
chimeric, bispecific or multispecific.
58. The isolated antibody, or antigen-binding fragment thereof, of
claim 47, wherein the antigen-binding fragment thereof comprises a
Fab, Fab2, or scFv.
59. A pharmaceutical composition comprising the antibody, or
antigen-binding fragment thereof, of claim 47, and a
pharmaceutically acceptable carrier.
60. A pharmaceutical composition comprising the antibody, or
antigen-binding fragment thereof, of claim 48, and a
pharmaceutically acceptable carrier.
61. A pharmaceutical composition comprising the antibody, or
antigen-binding fragment thereof, of claim 49, and a
pharmaceutically acceptable carrier
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Application No. 63/008,545, filed on Apr. 10, 2020, to
U.S. Provisional Application No. 63/021,589, filed on May 7, 2020,
to U.S. Provisional Application No. 63/046,313, filed on Jun. 30,
2020, to U.S. Provisional Application No. 63/112,122, filed on Nov.
10, 2020, to U.S. Provisional Application No. 63/138,886, filed on
Jan. 19, 2021, to U.S. Provisional Application No. 63/143,456,
filed on Jan. 29, 2021, to U.S. Provisional Application No.
63/147,495, filed on Feb. 9, 2021, to U.S. Provisional Application
No. 63/148,754, filed on Feb. 12, 2021, to U.S. Provisional
Application No. 63/150,413, filed on Feb. 17, 2021, to U.S.
Provisional Application No. 63/152,054, filed on Feb. 22, 2021, and
U.S. Provisional Application No. 63/163,400, filed on Mar. 19,
2021. The entire contents of each of these applications are
incorporated herein by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Apr. 8, 2021, is named 132280-00113_SL.txt and is 2,710,434
bytes in size.
FIELD
[0003] This disclosure generally pertains to antibodies and
antigen-binding fragments thereof, preferably human antibodies and
antigen-binding fragments and/or affinity-matured variants thereof,
recombinant cells engineered to express such antibodies, and
compositions containing such antibodies and antigen-binding
fragments thereof, wherein such antibodies and antigen-binding
fragments thereof specifically bind to the S protein of
coronaviruses ("CoV-S") and therapeutic and diagnostic uses for the
antibodies, antigen-binding fragments, and compositions
thereof.
BACKGROUND
[0004] Coronaviruses ("CoV") are genetically classified into four
major genera: the Alphacoronavirus genus (ACoV genus); the
Betacoronavirus genus (BCoV genus); the Gammacoronavirus genus
(CCoV genus); and Deltacoronavirus genus (DCoV genus), and while
ACoV and BCoV primarily infect mammals CCoV and DCoV predominantly
infect birds (Wu A. et al., Cell Host Microbe. 2020 Mar. 11;
27(3):325-328). Coronaviruses that infect humans were first
identified in the mid-1960s, and currently, seven confirmed CoV
species are known as human pathogens. Four CoV species, the
HCoV-HKU1 and HCoV-OC43 from the BCoV genus and the HCoV-229E and
HCoV-NL63 from the ACoV genus, are endemic species in humans and
cause mild respiratory symptoms, mostly in pediatric patients
(Brielle E. S., et al., BioRxiv reprint, doi:
https://doi.org/10.1101/2020.03.10.986398). The other three human
CoV species, the SARS-CoV, the MERS-CoV, and the SARS-CoV-2 (also
known as "2019-nCoV"), all of which are from the BCoV genus, have
caused severe outbreaks, including the Severe Acute Respiratory
Syndrome (SARS) outbreak in 2002-2003, the Middle East Respiratory
Syndrome (MERS) outbreak in 2012-2013, and the current (2019-)
pandemic of the coronavirus disease of 2019 ("COVID-19").
[0005] The genome of coronaviruses, whose size ranges between
approximately 26,000 and 32,000 bases, includes a variable number
(from 6 to 11) of open reading frames ("ORFs") (Wu A. et al., Cell
Host Microbe. 2020 Mar. 11; 27(3):325-328). The first ORF encodes
16 non-structural proteins ("nsps"), and the remaining ORFs encode
accessory proteins and structural proteins. The four major
structural proteins are the spike surface glycoprotein ("S protein"
or "S" or "spike protein"), small envelope protein ("E protein" or
"E"), matrix protein ("M protein" or "M"), and nucleocapsid protein
("N protein", or "N").
[0006] The S protein, which plays an essential role in binding to
receptors on the host cell and determines host tropism (Zhu Z. et
al., Infect Genet Evol. 2018 July; 61:183-184), forms homotrimers
protruding from the viral surface (Li F. Annu Rev Virol. 2016 Sep.
29; 3(1):237-261). The S protein is processed into two
non-covalently associated subunits, S1 and S2, and each monomer in
the trimeric S assembly is a heterodimer of S1 and S2 subunits.
Cryo-EM studies have revealed that the S1 subunit is comprised of
four domains: an N-terminal domain (NTD), a C-terminal domain
(CTD), and two subdomains (Walls A. C. et al., Nature 531, 114-117
(2016).; Tortorici M. A. and Veesler D., Adv Virus Res. 2019;
105:93-116; Wrapp D. et al., Science 367, 1260-1263 (2020)). The
CTD functions as the receptor-binding domain (RBD) for both
SARS-CoV and SARS-CoV-2 (Li F. J Virol. 2015 February;
89(4):1954-64). The S2 subunit contains the fusion peptide, heptad
repeat 1 and 2, and a transmembrane domain, all of which are
required to mediate fusion of the viral and host cell
membranes.
[0007] SARS-CoV and SARS-CoV-2 bind to and use
angiotensin-converting enzyme 2 (ACE2) of a host cell as a receptor
to enter the host cells (Ge X. Y. et al., Nature. 2013 Nov. 28;
503(7477):535-8; Hoffmann M. et al., Cell. 2020 Mar. 4. pii:
S0092-8674(20)30229-4). The motif within the RBD that particularly
binds to RCE2 is often referred to as the "ACE2-binding motif".
SARS-CoV can also use CD209L (also known as L-SIGN) as an
alternative receptor (Jeffers S. A. et al., Proc Natl Acad Sci USA.
2004 Nov. 2; 101(44):15748-53). In contrast, MERS-CoV binds
dipeptidyl peptidase 4 ("DPP4", also known as CD26) of the host
cell via a different RBD of the S protein.
[0008] Cell entry of coronaviruses often depends also on priming of
the S protein by host cell proteases. Recently, SARS-CoV-2 was
found to use the serine protease TMPRSS2 for S protein priming and
ACE2 for entry (Wu A. et al., Cell Host Microbe. 2020 Mar. 11;
27(3):325-328; Hoffmann M. et al., Cell. 2020 Mar. 4. pii:
S0092-8674(20)30229-4).
[0009] The genome of SARS-CoV-2 is about 29.8 kb nucleotides and
encodes 15 nsps, four structural proteins (S, E, M, and N) and
eight accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b, and orf14)
(Wu A. et al., Cell Host Microbe. 2020 Mar. 11; 27(3):325-328).
While SARS-CoV-2 is genetically close to a SARS-like bat CoV and
also to SARS-CoV, a number of sequence differences have been
identified. When SARS-CoV-2 is compared to SARS-CoV or SARS-like
bat CoV, 380 amino acid differences or substitutions were found, 27
of which are in the S protein, including 6 substitutions in the RBD
at amino acid region 357-528 (but not in the receptor-binding
motifs that directly interact with ACE2) and 6 substitutions in the
underpinning subdomain (SD) at amino acid region 569-655.
[0010] One of the few drugs approved by the U.S. Food and Drug
Administration ("FDA") for use in treating COVID-19 is the viral
replication inhibitor remdesivir. Clinical trials demonstrated that
remdesivir shortens the time to recovery in hospitalized patients,
but more effective therapy is in great need. Convalescent plasma
received the emergency use authorization status by the FDA. Other
treatments given to COVID-19 patients include anti-inflammatories
such as corticosteroids and other treatments for managing symptoms
such as supplemental oxygen and mechanical ventilatory support.
Several drugs, particularly those that have been approved for
preventing or treating other infectious disease, are currently
being tested in the clinic, which includes e.g.,
lopinavir-ritonavir (HIV protease inhibitor), ABX464 (viral RNA
splicer), favilavir (RNA-dependent RNA polymerase inhibitor used
for influenza virus infection), niclosamide and ivermectin
(antihelmintic), and BCG vaccine (vaccine for tuberculosis). Also,
other ongoing clinical trials reportedly are using IL-6 receptor
antagonist antibodies, an anti-GM-CSF or anti-GM-CSF receptor
antibody, an anti-TNF antibody, an anti-IL-1beta antibody, or an
anti-complement component 5 antibody, in an effort to inhibit
inflammation and thereby potentially inhibit cytokine storm and
sepsis which can manifest in some SARS-CoV-2-infected patients and
may cause death.
SUMMARY
[0011] In one aspect, the present disclosure relates to a compound
which binds to coronavirus (CoV) or the spike protein (S protein)
of a CoV ("CoV-S"). In some embodiments, the compound may be an
isolated antibody or antigen-binding antibody fragment which binds
to a CoV-S. In some embodiments, the antibody or antigen-binding
antibody fragment may comprise a heavy chain variable region (VH),
or fragments thereof, and/or a light chain variable region (VL), or
fragments thereof. In certain embodiments, the VH or fragment
thereof may comprise a complementarity-determining region 1 (CDR1),
a complementarity-determining region 2 (CDR2), and a
complementarity-determining region 3 (CDR3), which may also be
referred to as VH CDR1, VH CDR2, and VH CDR3, respectively. In
certain embodiments, the VL or fragment thereof may comprise a
CDR1, a CDR2, and a CDR3, which may also be referred to as VL CDR1,
VL CDR2, and VL CDR3, respectively. In some embodiments, the
antibody, or antigen-binding antibody fragment thereof, may
comprise a heavy chain CDR1, a heavy chain CDR2, a heavy chain
CDR3, a light chain CDR1, a light chain CDR2, and a light chain
CDR3.
[0012] In some embodiments, the antibody or antigen-binding
antibody fragment may comprise an antibody or antigen-binding
antibody fragment thereof, or an affinity-matured variant of an
anti-CoV-S antibody or antigen-binding antibody fragment thereof;
selected from the group consisting of ADI-55688, ADI-55689,
ADI-55690, ADI-55691, ADI-55692, ADI-55693, ADI-55694, ADI-55695,
ADI-55696, ADI-55697, ADI-55698, ADI-55699, ADI-55700, ADI-55701,
ADI-55702, ADI-55703, ADI-55704, ADI-55705, ADI-55706, ADI-55707,
ADI-55708, ADI-55709, ADI-55710, ADI-55711, ADI-55712, ADI-55713,
ADI-55714, ADI-55715, ADI-55716, ADI-55717, ADI-55718, ADI-55719,
ADI-55721, ADI-55722, ADI-55723, ADI-55724, ADI-55725, ADI-55726,
ADI-55727, ADI-55728, ADI-55729, ADI-55730, ADI-55731, ADI-55732,
ADI-55733, ADI-55734, ADI-55735, ADI-55736, ADI-55737, ADI-55738,
ADI-55739, ADI-55740, ADI-55741, ADI-55742, ADI-55743, ADI-55744,
ADI-55745, ADI-55746, ADI-55747, ADI-55748, ADI-55749, ADI-55750,
ADI-55751, ADI-55752, ADI-55753, ADI-55754, ADI-55755, ADI-55756,
ADI-55757, ADI-55758, ADI-55720, ADI-55760, ADI-55761, ADI-55762,
ADI-55763, ADI-55765, ADI-55766, ADI-55767, ADI-55769, ADI-55770,
ADI-55771, ADI-55775, ADI-55776, ADI-55777, ADI-55950, ADI-55951,
ADI-55952, ADI-55953, ADI-55954, ADI-55955, ADI-55956, ADI-55957,
ADI-55958, ADI-55959, ADI-55960, ADI-55961, ADI-55962, ADI-55963,
ADI-55964, ADI-55965, ADI-55966, ADI-55967, ADI-55968, ADI-55969,
ADI-55970, ADI-55972, ADI-55973, ADI-55974, ADI-55975, ADI-55976,
ADI-55977, ADI-55978, ADI-55979, ADI-55980, ADI-55981 ADI-55982,
ADI-55984, ADI-55986, ADI-55988, ADI-55989, ADI-55990, ADI-55992,
ADI-55993, ADI-55994, ADI-55995, ADI-55996, ADI-55997, ADI-55998,
ADI-55999, ADI-56000, ADI-56001, ADI-56002, ADI-56003, ADI-56004,
ADI-56005 ADI-56006, ADI-56007, ADI-56008, ADI-56009, ADI-56010,
ADI-56011, ADI-56012, ADI-56013, ADI-56014, ADI-56015, ADI-56016,
ADI-56017, ADI-56018, ADI-56019, ADI-56020, ADI-56021, ADI-56022,
ADI-56023, ADI-56024, ADI-56025, ADI-56026, ADI-56027, ADI-56028,
ADI-56029, ADI-56030, ADI-56031, ADI-56032, ADI-56033, ADI-56034,
ADI-56035, ADI-56037, ADI-56038, ADI-56039, ADI-56040, ADI-56041,
ADI-56042, ADI-56043, ADI-56044, ADI-56045, ADI-56046, ADI-56047,
ADI-56048, ADI-56049, ADI-56050, ADI-56051, ADI-56052, ADI-56053,
ADI-56054, ADI-56055, ADI-56056, ADI-56057, ADI-56058, ADI-56059,
ADI-56061, ADI-56062, ADI-56063, ADI-56064, ADI-56065, ADI-56066,
ADI-56067, ADI-56068, ADI-56069, ADI-56070, ADI-56071, ADI-56072,
ADI-56073, ADI-56074, ADI-56075 ADI-56076, ADI-56078, ADI-56079,
ADI-56080, ADI-56081, ADI-56082, ADI-56083, ADI-56084, ADI-56443,
ADI-56479, ADI-58120, ADI-58121, ADI-58122, ADI-58123, ADI-58124,
ADI-58125, ADI-58126, ADI-58127, ADI-58128, ADI-58129, ADI-58130,
ADI-58131, ADI-58130_LCN30cQ, and ADI-59988, optionally wherein the
CoV-S is SARS-CoV-S or SARS-CoV-2-S.
[0013] In particular embodiments, the antibody or antigen-binding
antibody fragment may comprise ADI-55688, ADI-55689, ADI-55690,
ADI-55951, ADI-55993, ADI-56000, ADI-56010, ADI-56032, or
ADI-56046, or an antigen-binding antibody fragment thereof; or an
affinity-matured variant of an anti-CoV-S antibody selected from
the group consisting of ADI-55688, ADI-55689, ADI-55690, ADI-55951,
ADI-55993, ADI-56000, ADI-56010, ADI-56032, and ADI-56046.
[0014] In particular embodiments, the antibody or antigen-binding
antibody fragment may comprise ADI-55689, ADI-55688, or ADI-56046,
or an antigen-binding antibody fragment thereof, or an
affinity-matured variant of an anti-CoV-S antibody ADI-55689,
ADI-55688, or ADI-56046, or an antigen-binding antibody fragment
thereof.
[0015] In further particular embodiments, the affinity-matured
variant may be ADI-57983 (with primer mutation), ADI-57978 (with
primer mutation), ADI-56868 (with primer mutation), ADI-58120,
ADI-58121, ADI-58122, ADI-58123, ADI-58124, ADI-58125, ADI-58126,
ADI-58127, ADI-58128, ADI-58129, ADI-58130, ADI-58131,
ADI-58130_LCN30cQ, or ADI-59988.
[0016] In some embodiments, the antibody, or antigen-binding
antibody fragment thereof, may comprise a VH and/or VL. In certain
embodiments, the VH may comprise a CDR3 having an amino acid
sequence identical to the VH CDR3 of any one of anti-CoV-S
antibodies described herein and in FIGS. 1, 2 and 36, and
optionally, the VL CDR3 may comprise a CDR3 having an amino acid
sequence identical to the VL CDR3 of the same anti-CoV-S antibody
that the VH CDR3 is derived from, and the anti-CoV-S antibody may
be selected from any one of anti-CoV-S antibodies described herein
and in FIGS. 1, 2 and 36. Here, the CoV-S may be the spike protein
("S protein") of Severe Acute Respiratory Syndrome (SARS)
coronavirus ("SARS-CoV"), which may be referred to as "SARS-CoV-S",
or the S protein of SARS-CoV-2 (also known as "n2019-nCoV"), which
may be referred to as "SARS-CoV-2-S". Optionally, the CoV-S may
comprise a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% identity to, comprising, or consisting of the
amino acid sequence of SEQ ID NO: 1 (SARS-CoV-S, 1288 amino acids,
Accession #PDB: 6VSB_B) or having at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% identity to, comprising, or consisting of
SEQ ID NO: 5 (SARS-CoV-2-S, 1273 amino acids, GenBank:
QHD43416.1).
[0017] In some preferred embodiments, the VH comprises a CDR3
having an amino acid sequence identical to the VH CDR3 of an
anti-CoV-S antibody selected from the group consisting of
ADI-55688, ADI-55689, ADI-55690, ADI-55951, ADI-55993, ADI-56000,
ADI-56010, ADI-56032, ADI-56046, ADI-57983 (with primer mutation),
ADI-57978 (with primer mutation), ADI-56868 (with primer mutation),
ADI-56443 (with primer mutation), ADI-56479 (with primer mutation),
ADI-58120, ADI-58121, ADI-58122, ADI-58123, ADI-58124, ADI-58125,
ADI-58126, ADI-58127, ADI-58128, ADI-58129, ADI-58130, ADI-58131,
ADI-58130_LCN30cQ, and ADI-59988. Optionally, the VL comprises a VL
CDR3 having an amino acid sequence identical to the CDR3 of the
same anti-CoV-S antibody from which the VH CDR3 is derived.
[0018] Further optionally, the isolated antibody or antigen-binding
antibody fragment may (a) cross-react with SARS-CoV-S and
SARS-CoV-2-S; and (b) may comprise the same VH CDR3 polypeptide
sequence as ADI-55708, ADI-55709, or ADI-55719, which is
ARGSLSREYDFLTAPQNGPWFDS (SEQ ID NO: 2108, 2208, or 3208); and
optionally (c) may further comprise the same VH CDR1 polypeptide
sequence as ADI-55708, ADI-55709, or ADI-55719VH.
[0019] In some embodiments, the antibody or antigen-binding
antibody fragment, optionally an affinity-matured variant of any of
the anti-CoV-S antibodies disclosed herein, may comprise at least
1, 2, 3, 4, 5 or all 6 complementarity-determining regions (CDRs)
of any one of anti-CoV-S antibodies described herein and in FIGS.
1, 2 and 36, optionally wherein the CoV-S is SARS-CoV-S or
SARS-CoV-2-S. Optionally, the CoV-S may comprise the amino acid
sequence of SEQ ID NO: 1 (SARS-CoV-S, 1288 amino acids, Accession
#PDB: 6VSB_B) or SEQ ID NO: 5 (SARS-CoV-2-S, 1273 amino acids,
GenBank: QHD43416.1).
[0020] In some preferred embodiments, the antibody or
antigen-binding antibody fragment comprises at least 1, 2, 3, 4, 5
or all 6 complementarity-determining regions (CDRs) of an
anti-CoV-S antibody selected from the group consisting of
ADI-55688, ADI-55689, ADI-55690, ADI-55951, ADI-55993, ADI-56000,
ADI-56010, ADI-56032, ADI-56046, ADI-57983 (with primer mutation),
ADI-57978 (with primer mutation), ADI-56868 (with primer mutation),
ADI-56443 (with primer mutation), ADI-56479 (with primer mutation),
ADI-58120, ADI-58121, ADI-58122, ADI-58123, ADI-58124, ADI-58125,
ADI-58126, ADI-58127, ADI-58128, ADI-58129, ADI-58130, ADI-58131,
ADI-58130_LCN30cQ, and ADI-59988.
[0021] In some embodiments, the isolated antibody or
antigen-binding antibody fragment, optionally an affinity-matured
variant of any of the anti-CoV-S antibodies disclosed herein, may
comprise: (a) a VH CDR1 polypeptide; (b) a VH CDR2 polypeptide; (c)
a VH CDR3 polypeptide; (d) a VL CDR1 polypeptide; (e) a VL CDR2
polypeptide; and (f) a VL CDR3 polypeptide. The amino acid
sequences of the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1,
the VL CDR2, and the VL CDR3 may be identical to the amino acid
sequences of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and
VL CDR3, respectively, of any one of anti-CoV-S antibodies
described herein and in FIGS. 1, 2 and 36. Optionally, the CoV-S
may be SARS-CoV-S or of "SARS-CoV-2-S". Further optionally, the
CoV-S may comprise the amino acid sequence of SEQ ID NO: 1
(SARS-CoV-S, 1288 amino acids, Accession #PDB: 6VSB_B) or SEQ ID
NO: 5 (SARS-CoV-2-S, 1273 amino acids, GenBank: QHD43416.1).
[0022] In particular embodiments, the amino acid sequences of the
VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2, and
the VL CDR3 may be identical to the amino acid sequences of the VH
CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3,
respectively, of an anti-CoV-S antibody selected from the group
consisting of ADI-55688, ADI-55689, ADI-55690, ADI-55951,
ADI-55993, ADI-56000, ADI-56010, ADI-56032, ADI-56046, ADI-57983
(with primer mutation), ADI-57978 (with primer mutation), ADI-56868
(with primer mutation), ADI-56443 (with primer mutation), ADI-56479
(with primer mutation), ADI-58120, ADI-58121, ADI-58122, ADI-58123,
ADI-58124, ADI-58125, ADI-58126, ADI-58127, ADI-58128, ADI-58129,
ADI-58130, ADI-58131, ADI-58130_LCN30cQ, and ADI-59988.
[0023] In further particular embodiments, the amino acid sequences
of the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2,
and the VL CDR3 may be identical to the amino acid sequences of the
VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3,
respectively, of an anti-CoV-S antibody selected from the group
consisting of ADI-57983 (with primer mutation), ADI-57978 (with
primer mutation), ADI-56868 (with primer mutation), ADI-56443 (with
primer mutation), ADI-56479 (with primer mutation), ADI-58120,
ADI-58121, ADI-58122, ADI-58123, ADI-58124, ADI-58125, ADI-58126,
ADI-58127, ADI-58128, ADI-58129, ADI-58130, ADI-58131,
ADI-58130_LCN30cQ, and ADI-59988.
[0024] In further preferred embodiments, the amino acid sequences
of the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2,
and the VL CDR3 may be identical to the amino acid sequences of the
VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3,
respectively, of an anti-CoV-S antibody selected from the group
consisting of ADI-57983 (with primer mutation), ADI-57978 (with
primer mutation), ADI-56868 (with primer mutation), ADI-58120,
ADI-58121, ADI-58122, ADI-58123, ADI-58124, ADI-58125, ADI-58126,
ADI-58127, ADI-58128, ADI-58129, ADI-58130, ADI-58131,
ADI-58130_LCN30cQ, and ADI-59988.
[0025] In certain embodiments, the isolated antibody or
antigen-binding antibody fragment, optionally an affinity-matured
variant of any of the anti-CoV-S antibodies disclosed herein, which
specifically binds to CoV-S, may comprise: (a) a VH comprising a VH
CDR1, VH CDR2, and VH CDR3; and (b) a VL comprising a VL CDR1, VL
CDR2, and VL CDR3.
[0026] In some exemplary embodiments, the amino acid sequences of
the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2,
and the VL CDR3 may be identical to the amino acid sequences
of:
(1) SEQ ID NOS: 104, 106, 108, 114, 116, and 118, respectively; (2)
SEQ ID NOS: 204, 206, 208, 214, 216, and 218, respectively; (3) SEQ
ID NOS: 304, 306, 308, 314, 316, and 318, respectively; (4) SEQ ID
NOS: 404, 406, 408, 414, 416, and 418, respectively; (5) SEQ ID
NOS: 504, 506, 508, 514, 516, and 518, respectively; (6) SEQ ID
NOS: 604, 606, 608, 614, 616, and 618, respectively; (7) SEQ ID
NOS: 704, 706, 708, 714, 716, and 718, respectively; (8) SEQ ID
NOS: 804, 806, 808, 814, 816, and 818, respectively; (9) SEQ ID
NOS: 904, 906, 908, 914, 916, and 918, respectively; (10) SEQ ID
NOS: 1004, 1006, 1008, 1014, 1016, and 1018, respectively; (11) SEQ
ID NOS: 1104, 1106, 1108, 1114, 1116, and 1118, respectively; (12)
SEQ ID NOS: 1204, 1206, 1208, 1214, 1216, and 1218, respectively;
(13) SEQ ID NOS: 1304, 1306, 1308, 1314, 1316, and 1318,
respectively; (14) SEQ ID NOS: 1404, 1406, 1408, 1414, 1416, and
1418, respectively; (15) SEQ ID NOS: 1504, 1506, 1508, 1514, 1516,
and 1518, respectively; (16) SEQ ID NOS: 1604, 1606, 1608, 1614,
1616, and 1618, respectively; (17) SEQ ID NOS: 1704, 1706, 1708,
1714, 1716, and 1718, respectively; (18) SEQ ID NOS: 1804, 1806,
1808, 1814, 1816, and 1818, respectively; (19) SEQ ID NOS: 1904,
1906, 1908, 1914, 1916, and 1918, respectively; (20) SEQ ID NOS:
2004, 2006, 2008, 2014, 2016, and 2018, respectively; (21) SEQ ID
NOS: 2104, 2106, 2108, 2114, 2116, and 2118, respectively; (22) SEQ
ID NOS: 2204, 2206, 2208, 2214, 2216, and 2218, respectively; (23)
SEQ ID NOS: 2304, 2306, 2308, 2314, 2316, and 2318, respectively;
(24) SEQ ID NOS: 2404, 2406, 2408, 2414, 2416, and 2418,
respectively; (25) SEQ ID NOS: 2504, 2506, 2508, 2514, 2516, and
2518, respectively; (26) SEQ ID NOS: 2604, 2606, 2608, 2614, 2616,
and 2618, respectively; (27) SEQ ID NOS: 2704, 2706, 2708, 2714,
2716, and 2718, respectively; (28) SEQ ID NOS: 2804, 2806, 2808,
2814, 2816, and 2818, respectively; (29) SEQ ID NOS: 2904, 2906,
2908, 2914, 2916, and 2918, respectively; (30) SEQ ID NOS: 3004,
3006, 3008, 3014, 3016, and 3018, respectively; (31) SEQ ID NOS:
3104, 3106, 3108, 3114, 3116, and 3118, respectively; (32) SEQ ID
NOS: 3204, 3206, 3208, 3214, 3216, and 3218, respectively; (33) SEQ
ID NOS: 3304, 3306, 3308, 3314, 3316, and 3318, respectively; (34)
SEQ ID NOS: 3404, 3406, 3408, 3414, 3416, and 3418, respectively;
(35) SEQ ID NOS: 3504, 3506, 3508, 3514, 3516, and 3518,
respectively; (36) SEQ ID NOS: 3604, 3606, 3608, 3614, 3616, and
3618, respectively; (37) SEQ ID NOS: 3704, 3706, 3708, 3714, 3716,
and 3718, respectively; (38) SEQ ID NOS: 3804, 3806, 3808, 3814,
3816, and 3818, respectively; (39) SEQ ID NOS: 3904, 3906, 3908,
3914, 3916, and 3918, respectively; (40) SEQ ID NOS: 4004, 4006,
4008, 4014, 4016, and 4018, respectively; (41) SEQ ID NOS: 4104,
4106, 4108, 4114, 4116, and 4118, respectively; (42) SEQ ID NOS:
4204, 4206, 4208, 4214, 4216, and 4218, respectively; (43) SEQ ID
NOS: 4304, 4306, 4308, 4314, 4316, and 4318, respectively; (44) SEQ
ID NOS: 4404, 4406, 4408, 4414, 4416, and 4418, respectively; (45)
SEQ ID NOS: 4504, 4506, 4508, 4514, 4516, and 4518, respectively;
(46) SEQ ID NOS: 4604, 4606, 4608, 4614, 4616, and 4618,
respectively; (47) SEQ ID NOS: 4704, 4706, 4708, 4714, 4716, and
4718, respectively; (48) SEQ ID NOS: 4804, 4806, 4808, 4814, 4816,
and 4818, respectively; (49) SEQ ID NOS: 4904, 4906, 4908, 4914,
4916, and 4918, respectively; (50) SEQ ID NOS: 5004, 5006, 5008,
5014, 5016, and 5018, respectively; (51) SEQ ID NOS: 5104, 5106,
5108, 5114, 5116, and 5118, respectively; (52) SEQ ID NOS: 5204,
5206, 5208, 5214, 5216, and 5218, respectively; (53) SEQ ID NOS:
5304, 5306, 5308, 5314, 5316, and 5318, respectively; (54) SEQ ID
NOS: 5404, 5406, 5408, 5414, 5416, and 5418, respectively; (55) SEQ
ID NOS: 5504, 5506, 5508, 5514, 5516, and 5518, respectively; (56)
SEQ ID NOS: 5604, 5606, 5608, 5614, 5616, and 5618, respectively;
(57) SEQ ID NOS: 5704, 5706, 5708, 5714, 5716, and 5718,
respectively; (58) SEQ ID NOS: 5804, 5806, 5808, 5814, 5816, and
5818, respectively; (59) SEQ ID NOS: 5904, 5906, 5908, 5914, 5916,
and 5918, respectively; (60) SEQ ID NOS: 6004, 6006, 6008, 6014,
6016, and 6018, respectively; (61) SEQ ID NOS: 6124, 6106, 6108,
6114, 6161, and 6118, respectively; (62) SEQ ID NOS: 6204, 6206,
6208, 6214, 6216, and 6218, respectively; (63) SEQ ID NOS: 6304,
6306, 6308, 6314, 6316, and 6318, respectively; (64) SEQ ID NOS:
6404, 6406, 6408, 6414, 6416, and 6418, respectively; (65) SEQ ID
NOS: 6504, 6506, 6508, 6514, 6516, and 6518, respectively; (66) SEQ
ID NOS: 6604, 6606, 6608, 6614, 6616, and 6618, respectively; (67)
SEQ ID NOS: 6704, 6706, 6708, 6714, 6716, and 6718, respectively;
(68) SEQ ID NOS: 6804, 6806, 6808, 6814, 6816, and 6818,
respectively; (69) SEQ ID NOS: 6904, 6906, 6908, 6914, 6916, and
6918, respectively; (70) SEQ ID NOS: 7004, 7006, 7008, 7014, 7016,
and 7018, respectively; (71) SEQ ID NOS: 7104, 7106, 7108, 7114,
7116, and 7118, respectively. (72) SEQ ID NOS: 7204, 7206, 7208,
7214, 7216, and 7218, respectively; (73) SEQ ID NOS: 7304, 7306,
7308, 7314, 7316, and 7318, respectively; (74) SEQ ID NOS: 7404,
7406, 7408, 7414, 7416, and 7418, respectively; (75) SEQ ID NOS:
7504, 7506, 7508, 7514, 7516, and 7518, respectively; (76) SEQ ID
NOS: 7604, 7606, 7608, 7614, 7616, and 7618, respectively; (77) SEQ
ID NOS: 7704, 7706, 7708, 7714, 7716, and 7718, respectively; (78)
SEQ ID NOS: 7804, 7806, 7808, 7814, 7816, and 7818, respectively;
(79) SEQ ID NOS: 7904, 7906, 7908, 7914, 7916, and 7918,
respectively; (80) SEQ ID NOS: 8004, 8006, 8008, 8014, 8016, and
8018, respectively; (81) SEQ ID NOS: 8104, 8106, 8108, 8114, 8116,
and 8118, respectively; (82) SEQ ID NOS: 8204, 8206, 8208, 8214,
8216, and 8218, respectively; (83) SEQ ID NOS: 8304, 8306, 8308,
8314, 8316, and 8318, respectively; (84) SEQ ID NOS: 8404, 8406,
8408, 8414, 8416, and 8418, respectively; (85) SEQ ID NOS: 8504,
8506, 8508, 8514, 8516, and 8518, respectively; (86) SEQ ID NOS:
8604, 8606, 8608, 8614, 8616, and 8618, respectively; (87) SEQ ID
NOS: 8704, 8706, 8708, 8714, 8716, and 8718, respectively; (88) SEQ
ID NOS: 8804, 8806, 8808, 8814, 8816, and 8818, respectively; (89)
SEQ ID NOS: 8904, 8906, 8908, 8914, 8916, and 8918, respectively;
(90) SEQ ID NOS: 9004, 9006, 9008, 9014, 9016, and 9018,
respectively; (101) SEQ ID NOS: 10104, 10106, 10108, 10114, 10116,
and 10118, respectively; (102) SEQ ID NOS: 10204, 10206, 10208,
10214, 10216, and 10218, respectively; (103) SEQ ID NOS: 10304,
10306, 10308, 10314, 10316, and 10318, respectively; (104) SEQ ID
NOS: 10404, 10406, 10408, 10414, 10416, and 10418, respectively;
(105) SEQ ID NOS: 10504, 10506, 10508, 10514, 10516, and 10518,
respectively; (106) SEQ ID NOS: 10604, 10606, 10608, 10614, 10616,
and 10618, respectively; (107) SEQ ID NOS: 10704, 10706, 10708,
10714, 10716, and 10718, respectively; (108) SEQ ID NOS: 10804,
10806, 10808, 10814, 10816, and 10818, respectively; (109) SEQ ID
NOS: 10904, 10906, 10908, 10914, 10916, and 10918, respectively;
(110) SEQ ID NOS: 11004, 11006, 11008, 11014, 11016, and 11018,
respectively; (111) SEQ ID NOS: 11104, 11106, 11108, 11114, 11116,
and 11118, respectively; (112) SEQ ID NOS: 11204, 11206, 11208,
11214, 11216, and 11218, respectively; (113) SEQ ID NOS: 11304,
11306, 11308, 11314, 11316, and 11318, respectively; (114) SEQ ID
NOS: 11404, 11406, 11408, 11414, 11416, and 11418, respectively;
(115) SEQ ID NOS: 11504, 11506, 11508, 11514, 11516, and 11518,
respectively; (116) SEQ ID NOS: 11604, 11606, 11608, 11614, 11616,
and 11618, respectively; (117) SEQ ID NOS: 11704, 11706, 11708,
11714, 11716, and 11718, respectively; (118) SEQ ID NOS: 11804,
11806, 11808, 11814, 11816, and 11818, respectively; (119) SEQ ID
NOS: 11904, 11906, 11908, 11914, 11916, and 11918, respectively;
(120) SEQ ID NOS: 12004, 12006, 12008, 12014, 12016, and 12018,
respectively; (121) SEQ ID NOS: 12104, 12106, 12108, 12114, 12116,
and 12118, respectively; (122) SEQ ID NOS: 12204, 12206, 12208,
12214, 12216, and 12218, respectively; (123) SEQ ID NOS: 12304,
12306, 12308, 12314, 12316, and 12318, respectively; (124) SEQ ID
NOS: 12404, 12406, 12408, 12414, 12416, and 12418, respectively;
(125) SEQ ID NOS: 12504, 12506, 12508, 12514, 12516, and 12518,
respectively; (126) SEQ ID NOS: 12604, 12606, 12608, 12614, 12616,
and 12618, respectively; (127) SEQ ID NOS: 12704, 12706, 12708,
12714, 12716, and 12718, respectively; (128) SEQ ID NOS: 12804,
12806, 12808, 12814, 12816, and 12818, respectively; (129) SEQ ID
NOS: 12904, 12906, 12908, 12914, 12916, and 12918, respectively;
(130) SEQ ID NOS: 13004, 13006, 13008, 13014, 13016, and 13018,
respectively; (131) SEQ ID NOS: 13104, 13106, 13108, 13114, 13116,
and 13118, respectively; (132) SEQ ID NOS: 13204, 13206, 13208,
13214, 13216, and 13218, respectively; (133) SEQ ID NOS: 13304,
13306, 13308, 13314, 13316, and 13318, respectively; (134) SEQ ID
NOS: 13404, 13406, 13408, 13414, 13416, and 13418, respectively;
(135) SEQ ID NOS: 13504, 13506, 13508, 13514, 13516, and 13518,
respectively; (136) SEQ ID NOS: 13604, 13606, 13608, 13614, 13616,
and 13618, respectively; (137) SEQ ID NOS: 13704, 13706, 13708,
13714, 13716, and 13718, respectively; (138) SEQ ID NOS: 13804,
13806, 13808, 13814, 13816, and 13818, respectively; (139) SEQ ID
NOS: 13904, 13906, 13908, 13914, 13916, and 13918, respectively;
(140) SEQ ID NOS: 14004, 14006, 14008, 14014, 14016, and 14018,
respectively; (141) SEQ ID NOS: 14104, 14106, 14108, 14114, 14116,
and 14118, respectively; (142) SEQ ID NOS: 14204, 14206, 14208,
14214, 14216, and 14218, respectively; (143) SEQ ID NOS: 14304,
14306, 14308, 14314, 14316, and 14318, respectively; (144) SEQ ID
NOS: 14404, 14406, 14408, 14414, 14416, and 14418, respectively;
(145) SEQ ID NOS: 14504, 14506, 14508, 14514, 14516, and 14518,
respectively; (146) SEQ ID NOS: 14604, 14606, 14608, 14614, 14616,
and 14618, respectively; (147) SEQ ID NOS: 14704, 14706, 14708,
14714, 14716, and 14718, respectively; (148) SEQ ID NOS: 14804,
14806, 14808, 14814, 14816, and 14818, respectively; (149) SEQ ID
NOS: 14904, 14906, 14908, 14914, 14916, and 14918, respectively;
(150) SEQ ID NOS: 15004, 15006, 15008, 15014, 15016, and 15018,
respectively; (151) SEQ ID NOS: 15104, 15106, 15108, 15114, 15116,
and 15118, respectively; (152) SEQ ID NOS: 15204, 15206, 15208,
15214, 15216, and 15218, respectively; (153) SEQ ID NOS: 15304,
15306, 15308, 15314, 15316, and 15318, respectively; (154) SEQ ID
NOS: 15404, 15406, 15408, 15414, 15416, and 15418, respectively;
(155) SEQ ID NOS: 15504, 15506, 15508, 15514, 15516, and 15518,
respectively; (156) SEQ ID NOS: 15604, 15606, 15608, 15614, 15616,
and 15618, respectively; (157) SEQ ID NOS: 15704, 15706, 15708,
15714, 15716, and 15718, respectively; (158) SEQ ID NOS: 15804,
15806, 15808, 15814, 15816, and 15818, respectively; (159) SEQ ID
NOS: 15904, 15906, 15908, 15914, 15916, and 15918, respectively;
(160) SEQ ID NOS: 16004, 16006, 16008, 16014, 16016, and 16018,
respectively; (161) SEQ ID NOS: 16104, 16106, 16108, 16114, 16116,
and 16118, respectively; (162) SEQ ID NOS: 16204, 16206, 16208,
16214, 16216, and 16218, respectively; (163) SEQ ID NOS: 16304,
16306, 16308, 16314, 16316, and 16318, respectively; (164) SEQ ID
NOS: 16404, 16406, 16408, 16414, 16416, and 16418, respectively;
(165) SEQ ID NOS: 16504, 16506, 16508, 16514, 16516, and 16518,
respectively; (166) SEQ ID NOS: 16604, 16606, 16608, 16614, 16616,
and 16618, respectively; (167) SEQ ID NOS: 16704, 16706, 16708,
16714, 16716, and 16718, respectively; (168) SEQ ID NOS: 16804,
16806, 16808, 16814, 16816, and 16818, respectively; (169) SEQ ID
NOS: 16904, 16906, 16908, 16914, 16916, and 16918, respectively;
(170) SEQ ID NOS: 17004, 17006, 17008, 17014, 17016, and 17018,
respectively; (171) SEQ ID NOS: 17104, 17106, 17108, 17114, 17116,
and 17118, respectively; (172) SEQ ID NOS: 17204, 17206, 17208,
17214, 17216, and 17218, respectively; (173) SEQ ID NOS: 17304,
17306, 17308, 17314, 17316, and 17318, respectively; (174) SEQ ID
NOS: 17404, 17406, 17408, 17414, 17416, and 17418, respectively;
(175) SEQ ID NOS: 17504, 17506, 17508, 17514, 17516, and 17518,
respectively; (176) SEQ ID NOS: 17604, 17606, 17608, 17614, 17616,
and 17618, respectively; (177) SEQ ID NOS: 17704, 17706, 17708,
17714, 17716, and 17718, respectively; (178) SEQ ID NOS: 17804,
17806, 17808, 17814, 17816, and 17818, respectively; (179) SEQ ID
NOS: 17904, 17906, 17908, 17914, 17916, and 17918, respectively;
(180) SEQ ID NOS: 18004, 18006, 18008, 18014, 18016, and 18018,
respectively; (181) SEQ ID NOS: 18104, 18106, 18108, 18114, 18116,
and 18118, respectively; (182) SEQ ID NOS: 18204, 18206, 18208,
18214, 18216, and 18218, respectively; (183) SEQ ID NOS: 18304,
18306, 18308, 18314, 18316, and 18318, respectively; (184) SEQ ID
NOS: 18404, 18406, 18408, 18414, 18416, and 18418, respectively;
(185) SEQ ID NOS: 18504, 18506, 18508, 18514, 18516, and 18518,
respectively; (186) SEQ ID NOS: 18604, 18606, 18608, 18614, 18616,
and 18618, respectively; (187) SEQ ID NOS: 18704, 18706, 18708,
18714, 18716, and 18718, respectively; (188) SEQ ID NOS: 18804,
18806, 18808, 18814, 18816, and 18818, respectively; (189) SEQ ID
NOS: 18904, 18906, 18908, 18914, 18916, and 18918, respectively;
(190) SEQ ID NOS: 19004, 19006, 19008, 19014, 19016, and 19018,
respectively; (191) SEQ ID NOS: 19104, 19106, 19108, 19114, 19116,
and 19118, respectively; (192) SEQ ID NOS: 19204, 19206, 19208,
19214, 19216, and 19218, respectively; (193) SEQ ID NOS: 19304,
19306, 19308, 19314, 19316, and 19318, respectively; (194) SEQ ID
NOS: 19404, 19406, 19408, 19414, 19416, and 19418, respectively;
(195) SEQ ID NOS: 19504, 19506, 19508, 19514, 19516, and 19518,
respectively; (196) SEQ ID NOS: 19604, 19606, 19608, 19614, 19616,
and 19618, respectively; (197) SEQ ID NOS: 19704, 19706, 19708,
19714, 19716, and 19718, respectively; (198) SEQ ID NOS: 19804,
19806, 19808, 19814, 19816, and 19818, respectively; (199) SEQ ID
NOS: 19904, 19906, 19908, 19914, 19916, and 19918, respectively;
(200) SEQ ID NOS: 20004, 20006, 20008, 20014, 20016, and 20018,
respectively; (201) SEQ ID NOS: 20104, 20106, 20108, 20114, 20116,
and 20118, respectively; (202) SEQ ID NOS: 20204, 20206, 20208,
20214, 20216, and 20218, respectively; (203) SEQ ID NOS: 20304,
20306, 20308, 20314, 20316, and 20318, respectively; (204) SEQ ID
NOS: 20404, 20406, 20408, 20414, 20416, and 20418, respectively;
(205) SEQ ID NOS: 20504, 20506, 20508, 20514, 20516, and 20518,
respectively; (206) SEQ ID NOS: 20604, 20606, 20608, 20614, 20616,
and 20618, respectively; (207) SEQ ID NOS: 20704, 20706, 20708,
20714, 20716, and 20718, respectively; (208) SEQ ID NOS: 20804,
20806, 20808, 20814, 20816, and 20818, respectively; (209) SEQ ID
NOS: 20904, 20906, 20908, 20914, 20916, and 20918, respectively;
(210) SEQ ID NOS: 21004, 21006, 21008, 21014, 21016, and 21018,
respectively; (211) SEQ ID NOS: 21104, 21106, 21108, 21114, 21116,
and 21118, respectively; (212) SEQ ID NOS: 21204, 21206, 21208,
21214, 21216, and 21218, respectively; (213) SEQ ID NOS: 21304,
21306, 21308, 21314, 21316, and 21318, respectively; (214) SEQ ID
NOS: 21404, 21406, 21408, 21414, 21416, and 21418, respectively;
(215) SEQ ID NOS: 21504, 21506, 21508, 21514, 21516, and 21518,
respectively; (216) SEQ ID NOS: 21604, 21606, 21608, 21614, 21616,
and 21618, respectively; (217) SEQ ID NOS: 21704, 21706, 21708,
21714, 21716, and 21718, respectively; (218) SEQ ID NOS: 21804,
21806, 21808, 21814, 21816, and 21818, respectively; (219) SEQ ID
NOS: 21904, 21906, 21908, 21914, 21916, and 21918, respectively;
(220) SEQ ID NOS: 22004, 22006, 22008, 22014, 22016, and 22018,
respectively; (221) SEQ ID NOS: 22104, 22106, 22108, 22114, 22116,
and 22118, respectively; (222) SEQ ID NOS: 22204, 22206, 22208,
22214, 22216, and 22218, respectively; (223) SEQ ID NOS: 22304,
22306, 22308, 22314, 22316, and 22318, respectively; (224) SEQ ID
NOS: 22404, 22406, 22408, 22414, 22416, and 22418, respectively;
(225) SEQ ID NOS: 22504, 22506, 22508, 22514, 22516, and 22518,
respectively; (226) SEQ ID NOS: 22604, 22606, 22608, 22614, 22616,
and 22618, respectively; (227) SEQ ID NOS: 22704, 22706, 22708,
22714, 22716, and 22718, respectively; (228) SEQ ID NOS: 22804,
22806, 22808, 22814, 22816, and 22818, respectively; (229) SEQ ID
NOS: 22904, 22906, 22908, 22914, 22916, and 22918, respectively;
(230) SEQ ID NOS: 23004, 23006, 23008, 23014, 23016, and 23018,
respectively; or (231) SEQ ID NOS: 23104, 23106, 23108, 23114,
23116, and 23118, respectively.
[0027] In particular exemplary embodiments, the amino acid
sequences of the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1,
the VL CDR2, and the VL CDR3 may be identical to the amino acid
sequences of:
(1) SEQ ID NOS: 104, 106, 108, 114, 116, and 118, respectively; (2)
SEQ ID NOS: 204, 206, 208, 214, 216, and 218, respectively; (3) SEQ
ID NOS: 304, 306, 308, 314, 316, and 318, respectively; (86) SEQ ID
NOS: 8604, 8606, 8608, 8614, 8616, and 8618, respectively; (123)
SEQ ID NOS: 12304, 12306, 12308, 12314, 12316, and 12318,
respectively; (130) SEQ ID NOS: 13004, 13006, 13008, 13014, 13016,
and 13018, respectively; (140) SEQ ID NOS: 14004, 14006, 14008,
14014, 14016, and 14018, respectively; (162) SEQ ID NOS: 16204,
16206, 16208, 16214, 16216, and 16218, respectively; (175) SEQ ID
NOS: 17504, 17506, 17508, 17514, 17516, and 17518, respectively;
(212) SEQ ID NOS: 21204, 21206, 21208, 21214, 21216, and 21218,
respectively; (213) SEQ ID NOS: 21304, 21306, 21308, 21314, 21316,
and 21318, respectively; (214) SEQ ID NOS: 21404, 21406, 21408,
21414, 21416, and 21418, respectively; (215) SEQ ID NOS: 21504,
21506, 21508, 21514, 21516, and 21518, respectively; (216) SEQ ID
NOS: 21604, 21606, 21608, 21614, 21616, and 21618, respectively;
(217) SEQ ID NOS: 21704, 21706, 21708, 21714, 21716, and 21718,
respectively; (218) SEQ ID NOS: 21804, 21806, 21808, 21814, 21816,
and 21818, respectively; (219) SEQ ID NOS: 21904, 21906, 21908,
21914, 21916, and 21918, respectively; (220) SEQ ID NOS: 22004,
22006, 22008, 22014, 22016, and 22018, respectively; (221) SEQ ID
NOS: 22104, 22106, 22108, 22114, 22116, and 22118, respectively;
(222) SEQ ID NOS: 22204, 22206, 22208, 22214, 22216, and 22218,
respectively; (223) SEQ ID NOS: 22304, 22306, 22308, 22314, 22316,
and 22318, respectively; (224) SEQ ID NOS: 22404, 22406, 22408,
22414, 22416, and 22418, respectively; (225) SEQ ID NOS: 22504,
22506, 22508, 22514, 22516, and 22518, respectively; (226) SEQ ID
NOS: 22604, 22606, 22608, 22614, 22616, and 22618, respectively; or
(227) SEQ ID NOS: 22704, 22706, 22708, 22714, 22716, and 22718,
respectively; (228) SEQ ID NOS: 22804, 22806, 22808, 22814, 22816,
and 22818, respectively; (229) SEQ ID NOS: 22904, 22906, 22908,
22914, 22916, and 22918, respectively; (230) SEQ ID NOS: 23004,
23006, 23008, 23014, 23016, and 23018, respectively; or (231) SEQ
ID NOS: 23104, 23106, 23108, 23114, 23116, and 23118,
respectively.
[0028] In further exemplary embodiments, the amino acid sequences
of the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2,
and the VL CDR3 may be identical to the amino acid sequences
of:
(212) SEQ ID NOS: 21204, 21206, 21208, 21214, 21216, and 21218,
respectively; (213) SEQ ID NOS: 21304, 21306, 21308, 21314, 21316,
and 21318, respectively; (214) SEQ ID NOS: 21404, 21406, 21408,
21414, 21416, and 21418, respectively; (215) SEQ ID NOS: 21504,
21506, 21508, 21514, 21516, and 21518, respectively; (216) SEQ ID
NOS: 21604, 21606, 21608, 21614, 21616, and 21618, respectively;
(217) SEQ ID NOS: 21704, 21706, 21708, 21714, 21716, and 21718,
respectively; (218) SEQ ID NOS: 21804, 21806, 21808, 21814, 21816,
and 21818, respectively; (219) SEQ ID NOS: 21904, 21906, 21908,
21914, 21916, and 21918, respectively; (220) SEQ ID NOS: 22004,
22006, 22008, 22014, 22016, and 22018, respectively; (221) SEQ ID
NOS: 22104, 22106, 22108, 22114, 22116, and 22118, respectively;
(222) SEQ ID NOS: 22204, 22206, 22208, 22214, 22216, and 22218,
respectively; (223) SEQ ID NOS: 22304, 22306, 22308, 22314, 22316,
and 22318, respectively; (224) SEQ ID NOS: 22404, 22406, 22408,
22414, 22416, and 22418, respectively; (225) SEQ ID NOS: 22504,
22506, 22508, 22514, 22516, and 22518, respectively; (226) SEQ ID
NOS: 22604, 22606, 22608, 22614, 22616, and 22618, respectively;
(227) SEQ ID NOS: 22704, 22706, 22708, 22714, 22716, and 22718,
respectively; (228) SEQ ID NOS: 22804, 22806, 22808, 22814, 22816,
and 22818, respectively; (229) SEQ ID NOS: 22904, 22906, 22908,
22914, 22916, and 22918, respectively; (230) SEQ ID NOS: 23004,
23006, 23008, 23014, 23016, and 23018, respectively; or (231) SEQ
ID NOS: 23104, 23106, 23108, 23114, 23116, and 23118,
respectively.
[0029] In other words, the amino acid sequences of the VH CDR1, the
VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 may
be identical to the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1,
the VL CDR2, and the VL CDR3 and VL amino acid sequences of any one
of anti-CoV-S antibodies described herein and in FIGS. 1, 2 and
36.
[0030] In some preferred embodiments, the amino acid sequences of
the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2,
and the VL CDR3 may be identical to the VH CDR1, the VH CDR2, the
VH CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 and VL amino
acid sequences of an antibody selected from the group consisting of
ADI-55688, ADI-55689, ADI-55690, ADI-55951, ADI-55993, ADI-56000,
ADI-56010, ADI-56032, ADI-56046, ADI-57983 (with primer mutation),
ADI-57978 (with primer mutation), ADI-56868 (with primer mutation),
ADI-56443 (with primer mutation), ADI-56479 (with primer mutation),
ADI-58120, ADI-58121, ADI-58122, ADI-58123, ADI-58124, ADI-58125,
ADI-58126, ADI-58127, ADI-58128, ADI-58129, ADI-58130, ADI-58131,
ADI-58130_LCN30cQ, and ADI-59988.
[0031] In further preferred embodiments, the amino acid sequences
of the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2,
and the VL CDR3 may be identical to the VH CDR1, the VH CDR2, the
VH CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 and VL amino
acid sequences of an antibody selected from the group consisting of
ADI-57983 (with primer mutation), ADI-57978 (with primer mutation),
ADI-56868 (with primer mutation), ADI-56443 (with primer mutation),
ADI-56479 (with primer mutation), ADI-58120, ADI-58121, ADI-58122,
ADI-58123, ADI-58124, ADI-58125, ADI-58126, ADI-58127, ADI-58128,
ADI-58129, ADI-58130, ADI-58131, ADI-58130_LCN30cQ, and
ADI-59988.
[0032] In some embodiments, the isolated antibody or
antigen-binding antibody fragment, optionally an affinity-matured
variant of any of the anti-CoV-S antibodies disclosed herein, may
possess one of the following structural features:
(1) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 102, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 112; (2) (a) the VH may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
202, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 212; (3) (a) the
VH may comprise an amino acid sequence with at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino
acid sequence of SEQ ID NO: 302, and (b) the VL may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 312; (4) (a) the VH may comprise an amino acid sequence with
at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 402, and (b) the
VL may comprise an amino acid sequence with at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino
acid sequence of SEQ ID NO: 412; (5) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 502, and (b) the VL may comprise an amino acid sequence with
at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 512; (6) (a) the
VH may comprise an amino acid sequence with at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino
acid sequence of SEQ ID NO: 602, and (b) the VL may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 612; (7) (a) the VH may comprise an amino acid sequence with
at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 702, and (b) the
VL may comprise an amino acid sequence with at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino
acid sequence of SEQ ID NO: 712; (8) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 802, and (b) the VL may comprise an amino acid sequence with
at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 812; (9) (a) the
VH may comprise an amino acid sequence with at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino
acid sequence of SEQ ID NO: 902, and (b) the VL may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 912; (10) (a) the VH may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 1002,
and (b) the VL may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 1012; (11) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 1102, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
1112; (12) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 1202, and (b) the
VL may comprise an amino acid sequence with at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino
acid sequence of SEQ ID NO: 1212; (13) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 1302, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 1312;
(14) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 1402, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 1412; (15) (a) the VH may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
1502, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 1512; (16) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 1602, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 1612; (17) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
1702, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 1712; (18) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 1802, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 1812; (19) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
1902, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 1912; (20) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 2002, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 2012; (21) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
2102, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 2112; (22) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 2202, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 2212; (23) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
2302, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 2312; (24) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 2402, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 2412; (25) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
2502, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 2512; (26) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 2602, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 2612; (27) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
2702, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 2712; (28) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 2802, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 2812; (29) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
2902, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 2912; (30) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 3002, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 3012; (31) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
3102, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 3112; (32) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 3202, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 3212; (33) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
3302, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 3312; (34) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 3402, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 3412; (35) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
3502, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 3512; (36) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 3602, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 3612; (37) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
3702, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 3712; (38) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 3802, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 3812; (39) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
3902, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 3912; (40) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 4002, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 4012; (41) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
4102, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 4112; (42) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 4202, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 4212; (43) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
4302, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 4312; (44) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 4402, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO:4412; (45) (a) the VH may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 4502,
and (b) the VL may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 4512; (46) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 4602, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
4612; (47) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 4702, and (b) the
VL may comprise an amino acid sequence with at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino
acid sequence of SEQ ID NO: 4712; (48) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 4802, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 4812;
(49) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 4902, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 4912; (50) (a) the VH may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
5002, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 5012; (51) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93,
94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 5102, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
5112; (52) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 5202, and (b) the
VL may comprise an amino acid sequence with at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino
acid sequence of SEQ ID NO: 5212; (53) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 5302, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 5312;
(54) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 5402, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 5412; (55) (a) the VH may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
5502, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 5512; (56) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 5602, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 5612; (57) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
5702, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 5712; (58) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 5802, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 5812; (59) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
5902, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 5912; (60) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 6002, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 6012; (61) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
6102, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 6112; (62) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 6202, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 6212; (63) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
6302, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 6312; (64) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 6402, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 6412; (65) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
6502, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 6512; (66) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 6602, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 6612; (67) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
6702, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 6712; (68) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 6802, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 6812; (69) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
6902, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 6912; (70) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 7002, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 7012; (71) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
7102, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 7112; (72) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 7202, and (b) the VL comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 7212; (73) (a) the VH may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 7302,
and (b) the VL may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 7312; (74) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 7402, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
7412; (75) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 7502, and (b) the
VL may comprise an amino acid sequence with at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino
acid sequence of SEQ ID NO: 7512; (76) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 7602, and (b) the VL comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 7612; (77) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 7702, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 7712; (78) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
7802, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 7812; (79) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 7902, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 7912; (80) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
8002, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 8012; (81) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 8102, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 8112; (82) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
8202, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 8212; (83) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 8302, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 8312; (84) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
8402, and (b) the VL nay comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 8412; (85) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 8502, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 8512; (86) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
8602, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 8612; (87) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 8702, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 8712; (88) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
8802, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 8812; (89) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 8902, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 8912; (90) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
9002, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 9012; (91) (a)
the VH may comprises an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 9102, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 9112; (92) (a) the VH may comprises an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
9202, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 9212; (93) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 9302, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 9312; (94) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
9402, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 9412; (95) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 9502, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 9512; (96) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
9602, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 9612; (97) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 9702, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 9712; (98) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
9802, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 9812; (99) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 9902, and (b) the VL may comprise
an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 9912; (100) (a) the VH may comprise an amino acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
10002, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 10012; (101) (a)
the VH may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid
sequence of SEQ ID NO: 10102, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
10112; (102) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 10202, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 10212; (103) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 10302, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
10312; (104) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 10402, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 10412; (105) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 10502, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
10512; (106) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 10602, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 10612; (107) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 10702, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
10712; (108) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 10802, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 10812; (109) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 10902, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
10912; (110) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 11002, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 11012; (111) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 11102, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
11112; (112) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 11202, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 11212; (113) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 11302, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
11312; (114) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 11402, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 11412; (115) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 11502, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
11512; (116) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 11602, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 11612; (117) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 11702, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
11712; (118) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 11802, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 11812; (119) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 11902, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
11912; (120) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 12002, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 12012; (121) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 12102, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
12112; (122) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 12202, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 12212; (123) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 12302, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
12312; (124) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 12402, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 12412; (125) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 12502, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
12512; (126) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 12602, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 12612; (127) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 12702, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
12712; (128) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 12802, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 12812; (129) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 12902, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
12912; (130) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 13002, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 13012; (131) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 13102, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
13112; (132) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 13202, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 13212; (133) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 13302, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
13312; (134) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 13402, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 13412; (135) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 13502, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
13512; (136) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 13602, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 13612; (137) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 13702, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
13712; (138) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 13802, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 13812; (139) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 13902, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
13912; (140) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 14002, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 14012; (141) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 14102, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
14112; (142) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 14202, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 14212; (143) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 14302, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
14312; (144) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 14402, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 14412; (145) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 14502, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
14512; (146) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 14602, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 14612; (147) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 14702, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
14712; (148) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 14802, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 14812; (149) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 14902, and (b) the VL comprises an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
14912; (150) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 15002, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 15012; (151) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 15102, and (b) the VL may comprise an amino
acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the amino acid sequence of SEQ ID NO:
15112; (152) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 15202, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 15212; (153) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 15302, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
15312; (154) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 15402, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 15412; (155) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 15502, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
15512; (156) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 15602, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 15612; (157) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 15702, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
15712; (158) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 15802, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 15812; (159) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 15902, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
15912; (160) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 16002, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 16012; (161) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 16102, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
16112; (162) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 16202, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 16212; (163) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 16302, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
16312; (164) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 16402, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 16412; (165) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 16502, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
16512; (166) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 16602, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 16612; (167) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 16702, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
16712; (168) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 16802, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 16812; (169) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 16902, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
16912; (170) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 17002, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 17012; (171) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 17102, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
17112; (172) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 17202, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 17212; (173) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 17302, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
17312; (174) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 17402, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 17412; (175) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 17502, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
17512; (176) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 17602, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 17612; (177) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 17702, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
17712; (178) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 17802, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 17812; (179) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 17902, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
17912; (180) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 18002, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 18012; (181) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 18102, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
18112; (182) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 18202, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 18212; (183) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 18302, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
18312; (184) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 18402, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 18412; (185) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 18502, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
18512; (186) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 18602, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 18612; (187) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 18702, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
18712; (188) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 18802, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 18812; (189) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 18902, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
18912; (190) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 19002, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 19012; (191) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 19102, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
19112; (192) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 19202, and (b)
the VL comprises an amino acid sequence with at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino
acid sequence of SEQ ID NO: 19212; (193) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 19302, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 19312;
(194) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 19402, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 19412; (195) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 19502, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 19512;
(196) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 19602, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 19612; (197) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 19702, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 19712;
(198) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 19802, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 19812; (199) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 19902, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 19912;
(200) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 20002, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 20012; (201) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 20102, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 20112;
(202) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 20202, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 20212; (203) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 20302, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 20312;
(204) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 20402, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 20412; (205) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 20502, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 20512;
(206) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 20602, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 20612; (207) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 20702, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 20712;
(208) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 20802, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 20812; (209) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 20902, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 20912;
(210) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 21002, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 21012; (211) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 21102, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 21112;
(212) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 21202, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 21212; (213) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 21302, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 21312;
(214) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 21402, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 21412; (215) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 21502, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 21512;
(216) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 21602, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 21612; (217) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 21702, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 21712;
(218) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 21802, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 21812; (219) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 21902, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 21912;
(220) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 22002, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 22012; or (221) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 22102, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 22112,
(222) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 22202, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 22212; (223) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 22302, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 22312;
(224) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 22402, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 22412; (225) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 22502, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 22512;
(226) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 22602, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 22612; or (227) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 22702, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 22712;
(228) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 22802, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 22812; (229) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 22902, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 22912;
(230) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 23002, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 23012; or (231) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 23102, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:
23112.
[0033] In some embodiments, the isolated antibody or
antigen-binding antibody fragment, optionally an affinity-matured
variant of any of the anti-CoV-S antibodies disclosed herein, may
possess one of the following structural features:
(1) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 102, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 112; (2) (a) the VH may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
202, and (b) the VL may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 212; (3) (a) the
VH may comprise an amino acid sequence with at least 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino
acid sequence of SEQ ID NO: 302, and (b) the VL may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 312; (86) (a) the VH may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 8602,
and (b) the VL may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 8612; (123) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 12302, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
12312; (130) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 13002, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 13012; (140) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 14002, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
14012; (162) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 16202, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 16212; (175) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 17502, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
17512.
[0034] In further embodiments:
(212) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 21202, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 21212; (213) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 21302, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 21312;
(214) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 21402, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 21412; (215) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 21502, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 21512;
(216) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 21602, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 21612; (217) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 21702, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 21712;
(218) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 21802, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 21812; (219) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 21902, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 21912;
(220) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 22002, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 22012; or (221) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 22102, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 22112,
(222) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 22202, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 22212; (223) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 22302, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 22312;
(224) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 22402, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 22412; (225) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 22502, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 22512;
(226) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 22602, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 22612; (227) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 22702, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 22712
(228) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 22802, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 22812; (229) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 22902, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 22912;
(230) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 23002, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 23012; or. (231) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 23102, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:
23112.
[0035] In further preferred embodiments:
(217) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 21702, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 21712; (218) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 21802, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 21812;
(219) (a) the VH may comprise an amino acid sequence with at least
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 21902, and (b) the VL may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 21912; (220) (a) the VH may comprise an
amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, or 100% sequence identity to the amino acid sequence of SEQ
ID NO: 22002, and (b) the VL may comprise an amino acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 22012;
or (221) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 22102, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 22112, (222) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 22202, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
22212; (223) (a) the VH may comprise an amino acid sequence with at
least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 22302, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 22312; (224) (a) the VH may
comprise an amino acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of SEQ ID NO: 22402, and (b) the VL may comprise an amino
acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
or 100% sequence identity to the amino acid sequence of SEQ ID NO:
22412; or (225) (a) the VH may comprise an amino acid sequence with
at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence
identity to the amino acid sequence of SEQ ID NO: 22502, and (b)
the VL may comprise an amino acid sequence with at least 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 22512.
[0036] In some embodiments, the present disclosure provides an
isolated antibody, or antigen-binding fragment thereof, which
specifically binds to the spike protein of a coronavirus ("CoV-S"),
wherein said antibody, or antigen-binding fragment thereof,
comprises at least one, e.g., two, three, four, five or six, of the
following sequences: [0037] (a) a heavy chain variable region (VH)
comprising a VH CDR1 having a sequence of
X.sub.1X.sub.2FX.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8 (SEQ ID
NO: 23205), wherein X.sub.1 is F or Y, X.sub.2 is T or S, X.sub.3
is S or T, X.sub.4 is S, R, G, H, T, A, or K, X.sub.5 Is F or Y,
X.sub.6 is Y, A, D, G, P, or V, X.sub.7 is M or I, and X.sub.8 is H
or N; [0038] (b) a heavy chain variable region (VH) comprising a VH
CDR1 having a sequence of X.sub.1TFX.sub.2SYYX.sub.3X.sub.4(SEQ ID
NO: 23206), wherein X.sub.1 is F or Y, X.sub.2 is T or S, X.sub.3
is M or I, and X.sub.4 is H or N; [0039] (c) a heavy chain variable
region (VH) comprising a VH CDR1 having a sequence of FTFSSYYMN
(SEQ ID NO: 23207); [0040] (d) a heavy chain variable region (VH)
comprising a VH CDR2 having a sequence of
SISX.sub.1DGYX.sub.2TYYPDSLKG (SEQ ID NO: 23208), wherein X.sub.1
is S or E and X.sub.2 is N or S; [0041] (e) a heavy chain variable
region (VH) comprising a VH CDR3 having a sequence of
ARDFSGHTAX.sub.1AGTGFEY (SEQ ID NO: 23209), wherein X.sub.1 is W or
V; [0042] (f) a light chain variable region (VL) comprising a VL
CDR1 having a sequence of TSGX.sub.1SNX.sub.2GAGYX.sub.3VH (SEQ ID
NO: 23210), wherein X.sub.1 is N or S, X.sub.2 is I or V, and
X.sub.3 is D or Y; [0043] (g) a light chain variable region (VL)
comprising a VL CDR2 having a sequence of
GX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6(SEQ ID NO: 23211),
wherein X.sub.1 is A, S, or T, X.sub.2 is S or T, X.sub.3 is S, T,
A, or N, X.sub.4 is R, L, or T, X.sub.5 is A, P, Q, H, or N, and
X.sub.6 is T or S; [0044] (h) a light chain variable region (VL)
comprising a VL CDR2 having a sequence of GX.sub.1SSX.sub.2X.sub.3S
(SEQ ID NO: 23212), wherein X.sub.1 is S or A, X.sub.2 is R or L,
and X.sub.3 is N, H, or Q; [0045] (i) a light chain variable region
(VL) comprising a VL CDR2 having a sequence of GSSSRNS (SEQ ID NO:
23213); and [0046] (j) a light chain variable region (VL)
comprising a VL CDR3 having a sequence of QSYDSSLSVLYX.sub.1 (SEQ
ID NO: 23214), wherein X.sub.1 is T or V.
[0047] In some embodiments, the antibody, or antigen-binding
fragment thereof, comprises a heavy chain variable region (VH)
comprising a VH CDR3 having a sequence of ARDFSGHTAX.sub.1AGTGFEY
(SEQ ID NO: 23209), wherein X.sub.1 is W or V, and a light chain
variable region (VL) comprising a VL CDR3 having a sequence of
QSYDSSLSVLYX.sub.1 (SEQ ID NO: 23214), wherein X.sub.1 is T or
V.
[0048] In other embodiments, the antibody, or antigen-binding
fragment thereof, comprises a heavy chain variable region (VH)
comprising a VH CDR3 having a sequence of ARDFSGHTAX.sub.1AGTGFEY
(SEQ ID NO: 23209), wherein X.sub.1 is W or V; a VH CDR2 having a
sequence of SISX.sub.1DGYX.sub.2TYYPDSLKG (SEQ ID NO: 23208),
wherein X.sub.1 is S or E and X.sub.2 is N or S; and a VH CDR1
having a sequence of X.sub.1TFX.sub.2SYYX.sub.3X.sub.4(SEQ ID NO:
23206), wherein X.sub.1 is F or Y, X.sub.2 is T or S, X.sub.3 is M
or I, and X.sub.4 is H or N; and a light chain variable region (VL)
comprising a VL CDR3 having a sequence of QSYDSSLSVLYX.sub.1 (SEQ
ID NO: 23214), wherein X.sub.1 is T or V; a VL CDR2 having a
sequence of GX.sub.1SSX.sub.2X.sub.3S (SEQ ID NO: 23212), wherein
X.sub.1 is S or A, X.sub.2 is R or L, and X.sub.3 is N, H, or Q;
and a VL CDR1 having a sequence of TSGX.sub.1SNX.sub.2GAGYX.sub.3VH
(SEQ ID NO: 23210), wherein X.sub.1 is N or S, X.sub.2 is I or V,
and X.sub.3 is D or Y.
[0049] In some embodiments, the present disclosure provides an
isolated antibody, or antigen-binding fragment thereof, which
specifically binds to the spike protein of a coronavirus ("CoV-S"),
wherein said antibody, or antigen-binding fragment thereof,
comprises at least one, e.g., two, three, four, five or six, of the
following sequences: [0050] (a) a heavy chain variable region (VH)
comprising a VH CDR1 having a sequence of
X.sub.1X.sub.2FX.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8 (SEQ ID
NO: 23205), wherein X.sub.1 is F or Y, X.sub.2 is T or S, X.sub.3
is S or T, X.sub.4 is S, R, G, H, T, A, or K, X.sub.5 Is F or Y,
X.sub.6 is Y, A, D, G, P, or V, X.sub.7 is M or I, and X.sub.8 is H
or N; [0051] (b) a heavy chain variable region (VH) comprising a VH
CDR1 having a sequence of FTFSX.sub.1X.sub.2AMH (SEQ ID NO: 23215),
wherein X.sub.1 is R or K and X.sub.2 is F or Y; [0052] (c) a heavy
chain variable region (VH) comprising a VH CDR1 having a sequence
of FTFSRFAMH (SEQ ID NO: 23216); [0053] (d) a heavy chain variable
region (VH) comprising a VH CDR2 having a sequence of
AINLNGDSX.sub.1YYTDSVRG (SEQ ID NO: 23217), wherein X.sub.1 is K or
T; [0054] (e) a heavy chain variable region (VH) comprising a VH
CDR3 having a sequence of VKDGGYYDSSGPGH (SEQ ID NO: 23218); [0055]
(f) a light chain variable region (VL) comprising a VL CDR1 having
a sequence of RASX.sub.1NX.sub.2X.sub.3X.sub.4X.sub.5LA (SEQ ID NO:
23219), wherein X.sub.1 is E or Q, X.sub.2 is I or V, X.sub.3 is L,
A, or N, X.sub.4 is H or N, and X.sub.5 is Y or W; [0056] (g) a
light chain variable region (VL) comprising a VL CDR1 having a
sequence of RASENIX.sub.1HYLA (SEQ ID NO: 23220), wherein X.sub.1
is L or A; [0057] (h) a light chain variable region (VL) comprising
a VL CDR1 having a sequence of RASENILHYLA (SEQ ID NO: 23221);
[0058] (i) a light chain variable region (VL) comprising a VL CDR2
having a sequence of
X.sub.1X.sub.2X.sub.3X.sub.4RX.sub.5X.sub.6(SEQ ID NO: 23222),
wherein X.sub.1 is D or E, X.sub.2 is A, S, T, or V, X.sub.3 is S
or F, X.sub.4 is S, A, T, K, N, or R; X.sub.5 is A or V, and
X.sub.6 is T, S, or P; [0059] (j) a light chain variable region
(VL) comprising a VL CDR2 having a sequence of DX.sub.1SX.sub.2RAT
(SEQ ID NO: 23223), wherein X.sub.1 is A, S, or V, X.sub.2 is K or
R; [0060] (k) a light chain variable region (VL) comprising a VL
CDR2 having a sequence of DASRRAT (SEQ ID NO: 23224); and [0061]
(l) a light chain variable region (VL) comprising a VL CDR3 having
a sequence of QQRX.sub.1NWPQN (SEQ ID NO: 23225), wherein X.sub.1
is A or S.
[0062] In some embodiments, the antibody, or antigen-binding
fragment thereof, comprises a heavy chain variable region (VH)
comprising a VH CDR3 having a sequence of VKDGGYYDSSGPGH (SEQ ID
NO: 23218), and a light chain variable region (VL) comprising a VL
CDR3 having a sequence of QQRX.sub.1NWPQN (SEQ ID NO: 23225),
wherein X.sub.1 is A or S.
[0063] In some embodiments, the antibody, or antigen-binding
fragment thereof, comprises a heavy chain variable region (VH)
comprising a VH CDR3 having a sequence of VKDGGYYDSSGPGH (SEQ ID
NO: 23218); a VH CDR2 having a sequence of AINLNGDSX.sub.1YYTDSVRG
(SEQ ID NO: 23217), wherein X.sub.1 is K or T; and a VH CDR1 having
a sequence of FTFS X.sub.1X.sub.2AMH (SEQ ID NO: 23215), wherein
X.sub.1 is R or K and X.sub.2 is F or Y; and a light chain variable
region (VL) comprising a VL CDR3 having a sequence of
QQRX.sub.1NWPQN (SEQ ID NO: 23225), wherein X.sub.1 is A or S; a VL
CDR2 having a sequence of DX.sub.1S X.sub.2RAT (SEQ ID NO: 23223),
wherein X.sub.1 is A, S, or V, X.sub.2 is K or R; and a VL CDR1
having a sequence of RASENIX.sub.1HYLA (SEQ ID NO: 23220), wherein
X.sub.1 is L or A.
[0064] In some embodiments, the present disclosure provides an
isolated antibody, or antigen-binding fragment thereof, which
specifically binds to the spike protein of a coronavirus ("CoV-S"),
wherein said antibody, or antigen-binding fragment thereof,
comprises at least one, e.g., two, three, four, five or six, of the
following sequences: [0065] (a) a heavy chain variable region (VH)
comprising a VH CDR1 having a sequence of FPFX.sub.1GTYMT (SEQ ID
NO: 23226), wherein X.sub.1 is K or S; [0066] (b) a heavy chain
variable region (VH) comprising a VH CDR1 having a sequence of
FX.sub.1FX.sub.2GX.sub.3X.sub.4MX.sub.5 (SEQ ID NO: 23227), wherein
X.sub.1 is P or I, X.sub.2 is K or S, X.sub.3 is T or H, X.sub.4 is
W or Y, and X.sub.5 is T or S; [0067] (c) a heavy chain variable
region (VH) comprising a VH CDR2 having a sequence of
IIYSGGDTYYADSVKG (SEQ ID NO: 23228); [0068] (d) a heavy chain
variable region (VH) comprising a VH CDR2 having a sequence of
X.sub.1X.sub.2YSGGX.sub.3TYYADX.sub.4VX.sub.5G (SEQ ID NO: 23229),
wherein X.sub.1 is V or I, X.sub.2 is L or I, X.sub.3 is D or S,
X.sub.4 is S or A, and X.sub.5 is K or Q; [0069] (e) a heavy chain
variable region (VH) comprising a VH CDR3 having a sequence of
ARDREMAIITERX.sub.1YGLDV (SEQ ID NO: 23230), wherein X.sub.1 is T
or S; [0070] (f) a light chain variable region (VL) comprising a VL
CDR1 having a sequence of SGGSSNIGSNSVN (SEQ ID NO: 23231); [0071]
(g) a light chain variable region (VL) comprising a VL CDR1 having
a sequence of SGX.sub.1X.sub.2X.sub.3NIX.sub.4SNX.sub.5VN (SEQ ID
NO: 23232), wherein X.sub.1 G or S, X.sub.2 is S or T, X.sub.3 is S
or A, X.sub.4 is G or A, and X.sub.5 is S or G; [0072] (h) a light
chain variable region (VL) comprising a VL CDR2 having a sequence
of X.sub.1X.sub.2SQRPS (SEQ ID NO: 23233), wherein X.sub.1 is S or
A and X.sub.2 is N or V; [0073] (i) a light chain variable region
(VL) comprising a VL CDR2 having a sequence of
X.sub.1X.sub.2X.sub.3X.sub.4RPS (SEQ ID NO: 23234), wherein X.sub.1
is G, S, or A, X.sub.2 is N or V, X.sub.3 is S or G, and X.sub.4 is
N or Q; and [0074] (j) a light chain variable region (VL)
comprising a VL CDR3 having a sequence of AAWDDSLNTFRYV (SEQ ID NO:
23235).
[0075] In some embodiments, the present disclosure provides an
isolated antibody, or antigen-binding fragment thereof, which
specifically binds to the spike protein of a coronavirus ("CoV-S"),
wherein said antibody, or antigen-binding fragment thereof,
comprises at least one, e.g., two, three, four, five or six, of the
following sequences: [0076] (a) a heavy chain variable region (VH)
comprising a VH CDR1 having a sequence of GTFSSX.sub.1AIS (SEQ ID
NO: 23236), wherein X.sub.1 is Y or D; [0077] (b) a heavy chain
variable region (VH) comprising a VH CDR1 having a sequence of
GX.sub.1FX.sub.2SX.sub.3X.sub.4X.sub.5X.sub.6(SEQ ID NO: 23237),
wherein X.sub.1 is T, S, or P, X.sub.2 is S or T, X.sub.3 is Y, F,
or D, X.sub.4 is V, G, or A, X.sub.5 is V, L, or I, and X.sub.6 is
S or T; [0078] (c) a heavy chain variable region (VH) comprising a
VH CDR2 having a sequence of GIIPIFVTANYAQKFQG (SEQ ID NO: 23238);
[0079] (d) a heavy chain variable region (VH) comprising a VH CDR2
having a sequence of
GIX.sub.1PIFX.sub.2TX.sub.3X.sub.4YAQKFQX.sub.5 (SEQ ID NO: 23239),
wherein X.sub.1 is I or M, X.sub.2 is G or V, X.sub.3 is A or T,
X.sub.4 is N or G, and X.sub.5 is G or D; [0080] (e) a heavy chain
variable region (VH) comprising a VH CDR3 having a sequence of
ARGRMATIRGGQNYYYYYGMDV (SEQ ID NO: 23240); [0081] (f) a light chain
variable region (VL) comprising a VL CDR1 having a sequence of
QASQDISNYLN (SEQ ID NO: 23241); [0082] (g) a light chain variable
region (VL) comprising a VL CDR1 having a sequence of
QASQDIX.sub.1X.sub.2X.sub.3LN (SEQ ID NO: 23242), wherein X.sub.1
is S or R, X.sub.2 is N or K, and X.sub.3 is Y or C; [0083] (h) a
light chain variable region (VL) comprising a VL CDR2 having a
sequence of DASNLET (SEQ ID NO: 23243); [0084] (i) a light chain
variable region (VL) comprising a VL CDR2 having a sequence of
X.sub.1X.sub.2SX.sub.3LX.sub.4X.sub.5 (SEQ ID NO: 23244), wherein
X.sub.1 is V, A, S, T, D, E, K, or R, X.sub.2 is A, V, or T,
X.sub.3 is S, N, T, or K, X.sub.4 is Q, E, K, or R, and X.sub.5 is
S, N, or T; [0085] (j) a light chain variable region (VL)
comprising a VL CDR3 having a sequence of QQYDNLPLT (SEQ ID NO:
23245); [0086] (k) a light chain variable region (VL) comprising a
VL CDR3 having a sequence of QQX.sub.1X.sub.2X.sub.3LPX.sub.4T (SEQ
ID NO: 23246), wherein X.sub.1 is Y or F, X.sub.2 is D or E,
X.sub.3 is N or D, and X.sub.4 is L or I; and [0087] (l) a light
chain variable region (VL) comprising a VL CDR3 having a sequence
of QX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5PX.sub.6X.sub.7(SEQ ID NO:
23247), wherein X.sub.1 is Q or H, X.sub.2 is Y, A, or F, X.sub.3
is D or E, X.sub.4 is N, S, or D, X.sub.5 is L, Y, or F, X.sub.6 is
I, L, T, V, or F, and X.sub.7 is T or A.
[0088] In some embodiments, the present disclosure provides an
isolated antibody, or antigen-binding fragment thereof, which
specifically binds to the spike protein of a coronavirus ("CoV-S"),
wherein said antibody, or antigen-binding fragment thereof,
comprises at least one, e.g., two, three, four, five or six, of the
following sequences: [0089] (a) a heavy chain variable region (VH)
comprising a VH CDR1 having a sequence of FTFDDYAMH (SEQ ID NO:
23248); [0090] (b) a heavy chain variable region (VH) comprising a
VH CDR1 having a sequence of FX.sub.1X.sub.2DDYAX.sub.3H (SEQ ID
NO: 23249), wherein X.sub.1 is T or I, X.sub.2 is F or L, and
X.sub.3 is M or V; [0091] (c) a heavy chain variable region (VH)
comprising a VH CDR2 having a sequence of
GISWNSGX.sub.1INYADSVX.sub.2G (SEQ ID NO: 23250), wherein X.sub.1
is T or S and X.sub.2 is M or K; [0092] (d) a heavy chain variable
region (VH) comprising a VH CDR2 having a sequence of
GIX.sub.1WNSGX.sub.2X.sub.3X.sub.4YADSVX.sub.5G (SEQ ID NO: 23251),
wherein X.sub.1 is S or T, X.sub.2 is S, T, or Y, X.sub.3 is I or
L, X.sub.4 is N or G, and X.sub.5 is K or M; [0093] (e) a heavy
chain variable region (VH) comprising a VH CDR3 having a sequence
of ASDSNYRDYYYHYGMDV (SEQ ID NO: 23252); [0094] (f) a light chain
variable region (VL) comprising a VL CDR1 having a sequence of
RSSQSLLHSXiGYNYLD (SEQ ID NO: 23253), wherein X.sub.1 is N or Q;
[0095] (g) a light chain variable region (VL) comprising a VL CDR2
having a sequence of LGSNRAS (SEQ ID NO: 23254); [0096] (h) a light
chain variable region (VL) comprising a VL CDR2 having a sequence
of X.sub.1X.sub.2X.sub.3X.sub.4RX.sub.5S (SEQ ID NO: 23255),
wherein X.sub.1 is L or G, X.sub.2 is G or N, X.sub.3 is S or N,
X.sub.4 is N or E, and X.sub.5 is A or S; [0097] (i) a light chain
variable region (VL) comprising a VL CDR3 having a sequence of
MQALQTPRT (SEQ ID NO: 23256); and [0098] (j) a light chain variable
region (VL) comprising a VL CDR3 having a sequence of
MQX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7T (SEQ ID NO:
23257), wherein X.sub.1 is A or S, X.sub.2 is L or I, X.sub.3 is Q
or no amino acid, X.sub.4 is Q or T, X.sub.5 is T, P, or V, X.sub.6
is P or G, and X.sub.7 is R, I, or V.
[0099] In some exemplary embodiments the isolated antibody or
antigen-binding antibody fragment, optionally an affinity-matured
variant, may be human, humanized, primatized or chimeric.
[0100] In some exemplary embodiments the isolated antibody or
antigen-binding antibody fragment, optionally an affinity-matured
variant, may be bispecific or multispecific.
[0101] In some exemplary embodiments the isolated antibody or
antigen-binding antibody fragment, optionally an affinity-matured
variant, may comprise at least one first antigen-binding domain
("ABD") and at least one second ABD.
[0102] Here, the following features (a) and (b) may be met:
(a) the first ABD may comprise the VH CDR1, the VH CDR2, the VH
CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 of a first
anti-CoV-S antibody selected from any one of anti-CoV-S antibodies
described herein and in FIGS. 1, 2 and 36; and/or (b) the second
ABD may comprise the VH CDR1, the VH CDR2, the VH CDR3, the VL
CDR1, the VL CDR2, and the VL CDR3 of a second anti-CoV-S antibody
selected from any one of anti-CoV-S antibodies described herein and
in FIGS. 1, 2 and 36.
[0103] Optionally, the first anti-CoV-S antibody may be same as the
second anti-CoV-S antibody or may be different from the second
anti-CoV-S antibody.
[0104] The first anti-CoV-S antibody and the second anti-CoV-S
antibody may bind to the same or different coronavirus species.
Optionally, the first CoV-S and the second CoV-S may be (i) both of
SARS-CoV or (ii) both of SARS-CoV-2.
[0105] Further optionally, the first anti-CoV-S antibody may be
same as the second anti-CoV-S antibody or may be different from the
second anti-CoV-S antibody. Still further optionally, these
antibodies may bind to the same or different epitopes on a CoV-S
expressed by said SARS-CoV or SARS-CoV-2. Alternatively, the first
anti-CoV-S antibody and the second anti-CoV-S antibody may bind to
different coronaviruses, optionally wherein the first CoV-S and the
second CoV-S are (i) SARS-CoV and of SARS-CoV-2 coronaviruses,
respectively, or are (ii) SARS-CoV-2 and of SARS-CoV coronaviruses,
respectively.
[0106] Furthermore, in some preferred embodiments, the following
features (a) and (b) may be met:
[0107] (a) the first ABD may comprise the VH CDR1, the VH CDR2, the
VH CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 of a first
anti-CoV-S antibody selected from the group consisting of
ADI-55688, ADI-55689, ADI-55690, ADI-55951, ADI-55993, ADI-56000,
ADI-56010, ADI-56032, ADI-56046, ADI-57983 (with primer mutation),
ADI-57978 (with primer mutation), ADI-56868 (with primer mutation),
ADI-56443 (with primer mutation), ADI-56479 (with primer mutation),
ADI-58120, ADI-58121, ADI-58122, ADI-58123, ADI-58124, ADI-58125,
ADI-58126, ADI-58127, ADI-58128, ADI-58129, ADI-58130, ADI-58131,
ADI-58130_LCN30cQ, and ADI-59988; and
[0108] (b) the second ABD may comprise the VH CDR1, the VH CDR2,
the VH CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 of a second
anti-CoV-S antibody selected from the group consisting of
ADI-55688, ADI-55689, ADI-55690, ADI-55951, ADI-55993, ADI-56000,
ADI-56010, ADI-56032, ADI-56046, ADI-57983 (with primer mutation),
ADI-57978 (with primer mutation), ADI-56868 (with primer mutation),
ADI-56443 (with primer mutation), ADI-56479 (with primer mutation),
ADI-57983, ADI-57978, ADI-56868, ADI-56443, and ADI-56479.
[0109] In some embodiments, the bispecific or multispecific
isolated antibody or antigen-binding antibody fragment may comprise
at least one first ABD and at least one second ABD.
[0110] In certain embodiments,
(a) the first ABD may comprise the VH CDR1, the VH CDR2, the VH
CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 of a first
anti-CoV-S antibody selected from any one of anti-CoV-S antibodies
described herein and in FIGS. 1, 2 and 36, or an affinity-matured
variant of any of the foregoing; and/or (b) the second ABD binds to
an antigen which may not be a CoV-S, optionally wherein the antigen
is a cytokine, a cytokine receptor, or an immunomodulatory
polypeptide. In certain preferred embodiments, in (a), the first
ABD may comprise the VH CDR1, the VH CDR2, the VH CDR3, the VL
CDR1, the VL CDR2, and the VL CDR3 of a first anti-CoV-S antibody
selected from the group consisting of ADI-55688, ADI-55689,
ADI-55690, ADI-55951, ADI-55993, ADI-56000, ADI-56010, ADI-56032,
ADI-56046, ADI-57983 (with primer mutation), ADI-57978 (with primer
mutation), ADI-56868 (with primer mutation), ADI-56443 (with primer
mutation), ADI-56479 (with primer mutation), ADI-57983, ADI-57978,
ADI-56868, ADI-56443, and ADI-56479, particularly preferably
selected from the group consisting of ADI-57983 (with primer
mutation), ADI-57978 (with primer mutation), ADI-56868 (with primer
mutation), ADI-56443 (with primer mutation), ADI-56479 (with primer
mutation), ADI-58120, ADI-58121, ADI-58122, ADI-58123, ADI-58124,
ADI-58125, ADI-58126, ADI-58127, ADI-58128, ADI-58129, ADI-58130,
ADI-58131, ADI-58130_LCN30cQ, and ADI-59988.
[0111] In some embodiments, the isolated antibody or
antigen-binding antibody fragment may comprise a Fab, Fab',
F(ab').sub.2, scFv, sc(Fv).sub.2, minibody, diabody, sdAb,
BITE.
[0112] In some embodiments, the isolated antibody or
antigen-binding antibody fragment may comprise a constant region or
Fc region or at least one domain thereof.
[0113] In certain embodiments, the constant region or Fc region may
comprise a mutation which impairs or enhances at least one effector
function, optionally FcR binding, FcRn binding, complement binding,
glycosylation, complement-dependent cytotoxicity ("CDC"), or
antibody-dependent cellular cytotoxicity ("ADCC").
[0114] In some embodiments, the constant or Fc region is primate
derived, preferably human.
[0115] The human constant or Fc region optionally may be selected
from a human IgG1, IgG2, IgG3 or IgG4 constant or Fc region which
optionally may be modified, optionally such as by domain deletion
or by introducing one or more mutations which impair or enhance at
least one effector function.
[0116] The present disclosure further relates to chimeric antigen
receptors ("CARs") comprising at least one antibody or
antigen-binding antibody fragment described herein.
[0117] The present disclosure further relates to antibody-drug
conjugates ("ADCs") comprising: (a) at least one antibody or
antigen-binding antibody fragment described herein; and (b) a
drug.
[0118] In some embodiments, the drug may be: (i) an antiviral drug,
which is optionally, remdesivir, favipiravir, darunavir,
nelfinavir, saquinavir, lopinavir or ritonavir; (ii) an
antihelminth drug, which may be optionally ivermectin; (iii) an
antiparasite drug, which may be optionally hydroxychloroquine,
chloroquine, or atovaquone; (iv) antibacterial vaccine, which may
be optionally the tuberculosis vaccine BCG; or (v) an
anti-inflammatory drug, which may be optionally a steroid such as
ciclesonide, a TNF inhibitor (e.g., adalimumab), a TNF receptor
inhibitor (e.g., etanercept), an IL-6 inhibitor (e.g.,
clazakizumab), an IL-6 receptor inhibitor (e.g., toclizumab), or
metamizole; (vi) an antihistamine drug, which may be optionally
bepotastine; (vii) an ACE inhibitor, which may be optionally
moexipril; (viii) a drug that inhibits priming of CoV-S, which may
be optionally a serine protease inhibitor such as nafamostat; or
(ix) a cytotoxic drug, which may be optionally daunorubicin,
mitoxantrone, doxorubicin, cucurbitacin, chaetocin, chaetoglobosin,
chlamydocin, calicheamicin, nemorubicin, cryptophyscin,
mensacarcin, ansamitocin, mitomycin C, geldanamycin,
mechercharmycin, rebeccamycin, safracin, okilactomycin, oligomycin,
actinomycin, sandramycin, hypothemycin, polyketomycin,
hydroxyellipticine, thiocolchicine, methotrexate, triptolide,
taltobulin, lactacystin, dolastatin, auristatin, monomethyl
auristatin E (MMAE), monomethyl auristatin F (MMAF), telomestatin,
tubastatin A, combretastatin, maytansinoid, MMAD, MMAF, DM1, DM4,
DTT, 16-GMB-APA-GA, 17-DMAP-GA, JW 55, pyrrolobenzodiazepine,
SN-38, Ro 5-3335, puwainaphycin, duocarmycin, bafilomycin, taxoid,
tubulysin, ferulenol, lusiol A, fumagillin, hygrolidin,
glucopiericidin, amanitin, ansatrienin, cinerubin, phallacidin,
phalloidin, phytosphongosine, piericidin, poronetin,
phodophyllotoxin, gramicidin A, sanguinarine, sinefungin,
herboxidiene, microcolin B, microcystin, muscotoxin A, tolytoxin,
tripolin A, myoseverin, mytoxin B, nocuolin A, psuedolaric acid B,
pseurotin A, cyclopamine, curvulin, colchicine, aphidicolin,
englerin, cordycepin, apoptolidin, epothilone A, limaquinone,
isatropolone, isofistularin, quinaldopeptin, ixabepilone,
aeroplysinin, arruginosin, agrochelin, or epothilone.
[0119] The present disclosure also relates to isolated nucleic
acids encoding any of the antibodies or antigen-binding antibody
fragments disclosed herein.
[0120] In some embodiments, the nucleic acid may comprise:
(212) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 21210, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 21220;
(213) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 21310, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 21320;
(214) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 21410, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 21420;
(215) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 21510, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 21520;
(216) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 21610, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 21620;
(217) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 21710, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 21720;
(218) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 21810, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 21820;
(219) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 21910, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 21920;
(220) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 22010, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 22020;
(221) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 22110, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 22120;
(222) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 22210, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 22220;
(223) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 22310, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 22320;
(224) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 22410, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 22420;
(225) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 22510, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 22520;
(226) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 22610, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 22620;
(227) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 22710, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 22720;
(228) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 22810, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 22820;
(229) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 22910, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 22920;
(230) (a) a nucleic acid sequence with at least 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO: 23010, and/or (b) a nucleic acid sequence
with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the nucleic acid sequence of SEQ ID NO: 23020;
or (231) (a) a nucleic acid sequence with at least 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, or 100% sequence identity to the nucleic
acid sequence of SEQ ID NO: 23110, and/or (b) a nucleic acid
sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100% sequence identity to the nucleic acid sequence of SEQ ID NO:
23120.
[0121] The present disclosure also relates to isolated cells which
may comprise any of the nucleic acids disclosed herein.
[0122] In some embodiments the cell may be a bacterial, yeast,
insect, fungal, or mammalian cell, optionally a human cell, further
optionally a CHO or HEK cell.
[0123] In some embodiments the cell may be a human immune cell,
optionally a T, NK, B or dendritic cell.
[0124] The present disclosure further relates to methods of
expressing the antibody or antigen-binding antibody fragment or the
CAR disclosed herein.
[0125] In some embodiments, the method may comprise: (a) culturing
the cell expressing an antibody or antigen-binding antibody
fragment or CAR of the present disclosure under conditions that
permit expression; and (b) optionally isolating the antibody or
antigen-binding antibody fragment or the CAR from the cell or the
culture medium containing the cell.
[0126] The present disclosure further relates to methods of
identifying an antibody or an antigen-binding antibody fragment
which specifically binds to CoV-S.
[0127] In some embodiments, the method may comprise: (a) obtaining
antisera and/or B cells obtained from a patient infected with
SARS-CoV or SARS-CoV-2, optionally wherein the patient recovered
from SARS-CoV or SARS-CoV-2 infection or the patient is a
convalescent patient infected with SARS-CoV or SARS-CoV-2; (b)
contacting the antisera and/or B cells with the CoV-S; and (c)
isolating an antibody or antigen-binding fragment thereof which
specifically bind to the CoV-S. Optionally, the CoV-S is the spike
protein of SARS-CoV ("SARS-CoV-S") or of SARS-CoV-2
("SARS-CoV-2-S"). Further optionally, the CoV-S may comprise the
amino acid sequence of SEQ ID NO: 1 (SARS-CoV-S, 1288 amino acids,
Accession #PDB: 6VSB_B) or SEQ ID NO: 5 (SARS-CoV-2-S, 1273 amino
acids, GenBank: QHD43416.1).
[0128] In some embodiments, the method may further detect that the
antibody or antigen-binding fragment thereof which specifically
binds to CoV-S neutralizes, blocks or inhibits coronavirus
infectivity or coronavirus proliferation, optionally wherein the
coronavirus is SARS-CoV or SARS-CoV-2.
[0129] In certain embodiments, the method may further detect
whether the antibody or antigen-binding antibody fragment thereof
which specifically binds to the CoV-S binds to other coronaviruses,
optionally selected from the group consisting of MERS-CoV,
HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63.
[0130] In any of such detection methods, the method may further
comprise determining the sequence of the antibody or
antigen-binding antibody fragment thereof may be determined.
[0131] In some embodiments these sequences may be affinity-matured
or mutated to enhance binding affinity and/or potentially increase
specificity to a particular CoV-S.
[0132] The present disclosure further provides compositions
comprising: (a) at least one antibody or antigen-binding antibody
fragment of the present disclosure; and (b) a pharmaceutically
acceptable carrier or excipient.
[0133] The present disclosure further provides methods of
determining whether a subject has been infected with SARS-CoV or
SARS-CoV-2 or another coronavirus by detecting whether a biological
sample from the subject may comprise SARS-CoV-S protein or
SARS-CoV-S-2 protein or another coronavirus S protein homologous
thereto based on its immunoreaction with at least one antibody or
antigen-binding antibody fragment disclosed herein. The sample may
optionally be blood, plasma, lymph, mucus, urine, and/or feces.
Optionally, the SARS-CoV S may comprise the amino acid sequence of
SEQ ID NO: 1 (SARS-CoV-S, 1288 amino acids, Accession #PDB:
6VSB_B),
[0134] Alternatively, the SARS-CoV-2 may comprise the amino acid
sequence of SEQ ID NO: 5 (SARS-CoV-2-S, 1273 amino acids, GenBank:
QHD43416.1).
[0135] Such determination methods optionally may comprise an ELISA
or radioimmunoassay.
[0136] In such determination methods, the subject optionally may be
human, a companion animal (e.g., a dog or cat), an agricultural
animal, e.g., animals used in meat production, or may comprise an
animal in a zoo, e.g., a tiger or lion.
[0137] In such determination methods, the samples optionally may be
collected at different times from the subject and the presence or
absence or the level of SARS-CoV-S or SARS-CoV-S-2 or another
coronavirus S protein homologous thereto may be detected in order
to assess whether the subject has recovered. Here, the SARS-CoV S
may comprise the amino acid sequence of SEQ ID NO: 1 (SARS-CoV-S,
1288 amino acids, Accession #PDB: 6VSB_B), and optionally the
SARS-CoV-2 may comprise the amino acid sequence of SEQ ID NO: 5
(SARS-CoV-2-S, 1273 amino acids, GenBank: QHD43416.1).
[0138] The present disclosure further provides methods of inducing
an immune response against SARS-CoV or SARS-CoV-2 or another
coronavirus, which may be selected from MERS-CoV, HCoV-HKU1,
HCoV-OC43, HCoV-229E, and HCoV-NL63, in a subject in need
thereof.
[0139] In some embodiments, the methods may comprise administering
at least one antibody or antigen-binding antibody fragment of the
present disclosure.
[0140] In some embodiments, the methods may comprise administering
a cocktail of different antibodies or antigen-binding antibody
fragments of the present disclosure, e.g., which bind to the same
or different epitopes on the same or different CoV-Ss.
[0141] In certain embodiments, the immune response elicits
immunoprotection, optionally prolonged, against at least one
coronavirus, optionally SARS-CoV or SARS-CoV-2, further optionally
against another coronavirus.
[0142] The present disclosure further provides methods of
inhibiting or blocking infection of susceptible cells by SARS-CoV
or SARS-CoV-2 or another coronavirus, such as MERS-CoV, HCoV-HKU1,
HCoV-OC43, HCoV-229E, and HCoV-NL63, in a subject in need
thereof.
[0143] In some embodiments, the method may comprise administering
at least one antibody or antigen-binding antibody fragment, of the
present disclosure, e.g., a cocktail as above-described.
[0144] The present disclosure further provides methods of treating
infection by SARS-CoV or SARS-CoV-2 or another coronavirus
optionally such as MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-229E, and
HCoV-NL63, or treating a condition, symptom, disease, or disorder
associated with said infection in a subject in need thereof.
[0145] In some embodiments, the method may comprise administering
to the subject a therapeutically effective amount of at least one
antibody or antigen-binding antibody fragment, an ADC or a CAR, of
the present disclosure, e.g., a cocktail as above-described.
[0146] In some embodiments, the condition, symptom, disease, or
disorder comprises at least one of bronchitis, pneumonia,
respiratory failure, acute respiratory failure, organ failure,
multi-organ system failure, pediatric inflammatory multisystem
syndrome, acute respiratory distress syndrome, blood clot, a
cardiac condition, myocardial injury, myocarditis, heart failure,
cardiac arrest, acute myocardial infarction, dysrhythmia, venous
thromboembolism, post-intensive care syndrome, shock, anaphylactic
shock, cytokine release syndrome, septic shock, disseminated
intravascular coagulation, ischemic stroke, intracerebral
hemorrhage, microangiopathic thrombosis, psychosis, seizure,
nonconvulsive status epilepticus, traumatic brain injury, stroke,
anoxic brain injury, encephalitis, posterior reversible
leukoencephalopathy, necrotizing encephalopathy, post-infectious
encephalitis, autoimmune mediated encephalitis, acute disseminated
encephalomyelitis, acute kidney injury, acute liver injury,
pancreatic injury, immune thrombocytopenia, subacute thyroiditis, a
gastrointestinal complication, aspergillosis, increased
susceptibility to infection with another virus or bacteria, and/or
a pregnancy-related complication.
[0147] The present disclosure also provides methods of preventing
infection by SARS-CoV or SARS-CoV-2 or another coronavirus
optionally selected from the group consisting of MERS-CoV,
HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 in a subject in need
thereof.
[0148] In some embodiments, the method may comprise administering
to the subject a prophylactically effective amount of at least one
antibody or antigen-binding antibody fragment, an ADC or a CAR, of
the present disclosure, e.g., a cocktail as above-described.
[0149] The present disclosure also provides methods of preventing
the need for a subject infected with SARS-CoV or SARS-CoV-2 or
another coronavirus optionally selected from the group consisting
of MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 to be
placed on a ventilator, or reducing the time that a subject
infected with SARS-CoV or SARS-CoV-2 or another coronavirus
optionally selected from the group consisting of MERS-CoV,
HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 is on a
ventilator.
[0150] In some embodiments, the method may comprise administering
to the subject a prophylactically or therapeutically effective
amount of at least one antibody or antigen-binding antibody
fragment, an ADC or a CAR, of the present disclosure, e.g., a
cocktail as above-described.
[0151] The present disclosure provides methods of preventing the
onset of pneumonia in a subject infected SARS-CoV or SARS-CoV-2 or
another coronavirus optionally selected from the group consisting
of MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63, or
treating pneumonia and/or the symptoms of pneumonia in a subject
for a subject infected SARS-CoV or SARS-CoV-2 or another
coronavirus optionally selected from the group consisting of
MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63.
[0152] In some embodiments, the method may comprise administering
to the subject a prophylactically or therapeutically effective
amount of at least one antibody or antigen-binding antibody
fragment, an ADC or a CAR, of the present disclosure, e.g., a
cocktail as above-described.
[0153] In any of such methods, the subject optionally may be human
or may comprise a companion animal, agricultural animal or animal
in a zoo.
[0154] Optionally the subject may have at least one risk factor
which renders them more prone to a poor clinical outcome.
[0155] In certain embodiments, wherein the risk factors may
comprise one or more of (i) advanced age such as over 55, 60 or 65
years old, (ii) diabetes, (iii) a chronic respiratory condition
such as asthma, cystic fibrosis, another fibrotic condition, or
COPD, (iv) obesity, (iv) hypertension, (v) a cardiac or
cardiovascular condition, such as heart defects or abnormalities,
(vi) a chronic inflammatory or autoimmune condition, e.g., lupus or
multiple sclerosis, and (vii) an immunocompromised status which
optionally may be caused by cancer, chemotherapy, smoking, bone
marrow or organ transplantation, immune deficiencies, poorly
controlled HIV infection or AIDS, or prolonged use of
corticosteroids or other immunosuppressive medications. In certain
embodiments, the subject may further be treated with at least one
other drug. In certain embodiments, the method further comprises
administering to the subject at least one other drug. Optionally,
such one other drug may be: (i) an antiviral drug, optionally,
remdesivir, favipiravir, darunavir, nelfinavir, saquinavir,
lopinavir, or ritonavir; (ii) an antihelminth drug, optionally
ivermectin; (iii) an antiparasitic drug, optionally
hydroxychloroquine, chloroquine, or atovaquone; (iv) antibacterial
vaccine, optionally the tuberculosis vaccine BCG; (v) an
anti-inflammatory drug, optionally a steroid such as ciclesonide, a
TNF inhibitor (e.g., adalimumab), a TNF receptor inhibitor (e.g.,
etanercept), an IL-6 inhibitor (e.g., clazakizumab), an IL-6
receptor inhibitor (e.g., tocilizumab), or metamizole; (vi) an
antihistamine drug, optionally bepotastine; (vii) an ACE inhibitor,
optionally moexipril; and/or (viii) a drug that inhibits priming of
CoV-S, optionally a serine protease inhibitor, further optionally
nafamostat.
[0156] In certain embodiments, the subject may further be treated
with: (I) an antiviral agent, optionally, remdesivir, favipiravir,
darunavir, nelfinavir, saquinavir, lopinavir, or ritonavir; and
(II) at least one other drug. In certain embodiments, the method
may further comprise administering to the subject (I) an antiviral
agent, optionally, remdesivir, favipiravir, darunavir, nelfinavir,
saquinavir, lopinavir, or ritonavir; and (II) at least one other
drug. Optionally, the at least one other drug may be (i) an
antihelminth drug, further optionally ivermectin; (ii) an
antiparasitic drug, optionally hydroxychloroquine, chloroquine, or
atovaquone; (iii) an antibacterial vaccine, which is optionally the
tuberculosis vaccine BCG; or (iv) an anti-inflammatory drug,
optionally a steroid such as ciclesonide, a TNF inhibitor (e.g.,
adalimumab), a TNF receptor inhibitor (e.g., etanercept), an IL-6
inhibitor (e.g., clazakizumab), an IL-6 receptor inhibitor (e.g.,
toclizumab), or metamizole; (v) an antihistamine drug, optionally
bepotastine; (vi) an ACE inhibitor, optionally moexipril; and/or
(vii) a drug that inhibits priming of CoV-S, which is optionally a
serine protease inhibitor such as nafamostat.
[0157] In some embodiments, anti-CoV-S antibodies or
antigen-binding antibody fragments of the present disclosure may be
characterized by having a certain VH CDR3 sequences or having a VH
CDR3 sequences that are similar to a certain VH CDR3.
[0158] In some embodiments, antibodies or antigen-binding antibody
fragments of the present may comprise: (i) a VH and a VL or (ii) a
VH, and the VH may comprise a CDR3 having the amino acid sequence
of SEQ ID NO: 2108, 21708, 22208, 22408, 22608, or 22708.
[0159] In certain embodiments, antibodies or antigen-binding
antibody fragments of the present may comprise a VH and a VL, and
the VH may additionally comprise a CDR1 and a CDR3 having the amino
acid sequence of SEQ ID NOS: 2104 and 2108, respectively, SEQ ID
NOS: 21704 and 21708, respectively, SEQ ID NOS: 22204 and 22208,
respectively, SEQ ID NOS: 22404 and 22408, respectively, SEQ ID
NOS: 22604 and 22608, respectively, or SEQ ID NOS: 22704 and 22708,
respectively.
[0160] In further embodiments, antibody or antigen-binding antibody
fragment of the present may comprise (I) a VH and a VL or (II) a
VH, and the amino acid sequence of the VH CDR3 may be: (i)
identical to the amino acid sequence of SEQ ID NO: 1508 or differ
from SEQ ID NO: 1508 by 1, 2, or 3 amino acids; (ii) identical to
the amino acid sequence of SEQ ID NO: 1308 or differ from SEQ ID
NO: 1308 by 1, 2, or 3 amino acids; (iii) identical to the amino
acid sequence of SEQ ID NO: 808 or differ from SEQ ID NO: 808 by 1,
2, or 3 amino acids; (iv) identical to the amino acid sequence of
SEQ ID NO: 108 or differ from SEQ ID NO: 108 by 1, 2, or 3 amino
acids; (v) identical to the amino acid sequence of SEQ ID NO: 2108
or differ from SEQ ID NO: 2108 by 1, 2, or 3 amino acids; (vi)
identical to the amino acid sequence of SEQ ID NO: 108 or differs
from SEQ ID NO: 108 by 1, 2, or 3 amino acids; (vii) identical to
the amino acid sequence of SEQ ID NO: 208 or differs from SEQ ID
NO: 208 by 1, 2, or 3 amino acids; (viii) identical to the amino
acid sequence of SEQ ID NO: 308 or differs from SEQ ID NO: 308 by
1, 2, or 3 amino acids; (ix) identical to the amino acid sequence
of SEQ ID NO: 8608 or differs from SEQ ID NO: 8608 by 1, 2, or 3
amino acids; (x) identical to the amino acid sequence of SEQ ID NO:
12308 or differs from SEQ ID NO: 12308 by 1, 2, or 3 amino acids;
(xi) identical to the amino acid sequence of SEQ ID NO: 13008 or
differs from SEQ ID NO: 13008 by 1, 2, or 3 amino acids; (xii)
identical to the amino acid sequence of SEQ ID NO: 14008 or differs
from SEQ ID NO: 14008 by 1, 2, or 3 amino acids; (xiii) identical
to the amino acid sequence of SEQ ID NO: 16208 or differs from SEQ
ID NO: 16208 by 1, 2, or 3 amino acids; (xiv) identical to the
amino acid sequence of SEQ ID NO: 17508 or differs from SEQ ID NO:
17508 by 1, 2, or 3 amino acids; (xv) identical to the amino acid
sequence of SEQ ID NO: 21708 or differs from SEQ ID NO: 21708 by 1,
2, or 3 amino acids; (xvi) identical to the amino acid sequence of
SEQ ID NO: 22208 or differs from SEQ ID NO: 22208 by 1, 2, or 3
amino acids; (xvii) identical to the amino acid sequence of SEQ ID
NO: 22408 or differs from SEQ ID NO: 22408 by 1, 2, or 3 amino
acids; (xviii) identical to the amino acid sequence of SEQ ID NO:
22608 or differs from SEQ ID NO: 22608 by 1, 2, or 3 amino acids;
or (xix) identical to the amino acid sequence of SEQ ID NO: 22708
or differs from SEQ ID NO: 22708 by 1, 2, or 3 amino acids; or (xx)
identical to the amino acid sequence of SEQ ID NO: 23008 or differs
from SEQ ID NO: 23008 by 1, 2, or 3 amino acids.
[0161] In one embodiment, the anti-CoV-S antibodies or
antigen-binding antibody fragment may comprise the VH CDR1, VH
CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, respectively, of an
anti-CoV-S antibody selected from the group consisting of
ADI-55688, ADI-55689, ADI-55690, ADI-55951, ADI-55993, ADI-56000,
ADI-56010, ADI-56032, ADI-56046, ADI-57983 (with primer mutation),
ADI-57978 (with primer mutation), ADI-56868 (with primer mutation),
ADI-56443 (with primer mutation), ADI-56479 (with primer mutation),
ADI-57983, ADI-57978, ADI-56868, ADI-56443, and ADI-56479,
optionally of an anti-CoV-S antibody selected from the group
consisting of ADI-57983 (with primer mutation), ADI-57978 (with
primer mutation), ADI-56868 (with primer mutation), ADI-56443 (with
primer mutation), ADI-56479 (with primer mutation), ADI-58120,
ADI-58121, ADI-58122, ADI-58123, ADI-58124, ADI-58125, ADI-58126,
ADI-58127, ADI-58128, ADI-58129, ADI-58130, ADI-58131,
ADI-58130_LCN30cQ, and ADI-59988, and further optionally of an
anti-CoV-S antibody selected from the group consisting of ADI-57983
(with primer mutation), ADI-57978 (with primer mutation), ADI-56868
(with primer mutation), ADI-58120, ADI-58121, ADI-58122, ADI-58123,
ADI-58124, ADI-58125, ADI-58126, ADI-58127, ADI-58128, ADI-58129,
ADI-58130, ADI-58131, ADI-58130_LCN30cQ, and ADI-59988.
[0162] In some embodiments, the antibody or antigen-binding
antibody fragment may specifically compete with another antibody or
antigen-binding antibody fragment that may comprise the VH CDR1, VH
CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, respectively, of an
anti-CoV-S antibody selected from the group consisting of
ADI-55688, ADI-55689, ADI-55690, ADI-55951, ADI-55993, ADI-56000,
ADI-56010, ADI-56032, ADI-56046, ADI-57983 (with primer mutation),
ADI-57978 (with primer mutation), ADI-56868 (with primer mutation),
ADI-56443 (with primer mutation), ADI-56479 (with primer mutation),
ADI-58120, ADI-58121, ADI-58122, ADI-58123, ADI-58124, ADI-58125,
ADI-58126, ADI-58127, ADI-58128, ADI-58129, ADI-58130, ADI-58131,
ADI-58130_LCN30cQ, or ADI-59988, and preferably of ADI-57983 (with
primer mutation), ADI-57978 (with primer mutation), ADI-56868 (with
primer mutation), ADI-56443 (with primer mutation), ADI-56479 (with
primer mutation), ADI-58120, ADI-58121, ADI-58122, ADI-58123,
ADI-58124, ADI-58125, ADI-58126, ADI-58127, ADI-58128, ADI-58129,
ADI-58130, ADI-58131, ADI-58130_LCN30cQ, or ADI-59988, optionally
of ADI-57983 (with primer mutation), ADI-57978 (with primer
mutation), ADI-56868 (with primer mutation), ADI-58120, ADI-58121,
ADI-58122, ADI-58123, ADI-58124, ADI-58125, ADI-58126, ADI-58127,
ADI-58128, ADI-58129, ADI-58130, ADI-58131, ADI-58130_LCN30cQ, and
ADI-59988.
[0163] In certain embodiments, antibody or antigen-binding antibody
fragment of the present disclosure may comprise an Fc region. The
Fc region may comprise a wild type sequence or a variant sequence
and optionally may comprise an amino acid sequence of SEQ ID NOs:
11, 12, 13, 14, 15, 16, or 17.
[0164] In certain embodiments, the isolated antibody or
antigen-binding antibody fragment may bind to the S1 subunit of
SARS-CoV-S or of SARS-CoV-2-S.
[0165] In certain embodiments, the isolated antibody or
antigen-binding antibody fragment may bind to the receptor binding
domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-S or of
SARS-CoV-2-S.
[0166] In certain embodiments, the isolated antibody or
antigen-binding antibody fragment may bind to the ACE2-binding
motif of SARS-CoV-S or of SARS-CoV-2-S and optionally further binds
to the epitope of the antibody CR3022.
[0167] In further embodiments, the isolated antibody or
antigen-binding antibody fragment may compete with ACE2.
[0168] In further embodiments, the isolated antibody or
antigen-binding antibody fragment may compete with: (i) ACE2 and
the antibody CR3022; or (ii) ACE2 but not the antibody CR3022. In
certain embodiments, the isolated antibody or antigen-binding
antibody fragment (a) may bind to the S protein of SARS-CoV and/or
of SARS-CoV-2; and (b) may not bind to any of the S proteins of
HCoV-229E, HCoV-HKU1, HCoV-NL63, and HCoV-OK43.
[0169] In certain embodiments, the isolated antibody or
antigen-binding antibody fragment may (a) bind to the S protein of
SARS-CoV and/or of SARS-CoV-2; and also (b) bind to the S protein
of at least one of HCoV-229E, HCoV-HKU1, HCoV-NL63, and
HCoV-OK43.
[0170] In further embodiments, the isolated antibody or
antigen-binding antibody fragment may neutralize SARS-CoV and/or
SARS-CoV-2.
[0171] In further embodiments, the isolated antibody or
antigen-binding antibody fragment may neutralize SARS-CoV and/or
SARS-CoV-2 at 100 nM in vitro.
[0172] In further embodiments, the isolated antibody or
antigen-binding antibody fragment may neutralize SARS-CoV and/or
SARS-CoV-2 at: (i) an IC50 of about 100 nM or lower, of about 50 nM
or lower, of about 20 nM or lower, of about 10 nM or lower, of
about 5 nM or lower, of about 2 nM or lower, of about 1 nM or
lower, of about 500 pM or lower, of about 200 pM or lower, of about
100 pM or lower, of about 50 pM or lower, of about 20 pM or lower,
of about 10 pM or lower, of about 5 pM or lower, of about 2 pM or
lower, or of about 1 pM or lower; and/or (ii) an IC50 of about 500
ng/mL or lower, of about 200 ng/mL or lower, of about 100 ng/mL or
lower, of about 50 ng/mL or lower, of about 20 ng/mL or lower, of
about 10 ng/mL or lower, of about 20 ng/mL or lower, of about 10
mg/mL or lower, of about 5 ng/mL or lower, of about 2 ng/mL or
lower, or of about 1 ng/mL or lower, in vitro, optionally as
measured by any of the neutralization assays described in the
Examples herein.
[0173] In further embodiments, the isolated antibody or
antigen-binding antibody fragment may bind to CoV-S(S protein of
any CoV, such as but not limited to SARS-CoV-S and/or SARS-CoV-2-S)
with a KD value of: (i) 100 nM or lower; (ii) 10 nM or lower; (iii)
1 nM or lower; (iv) 100 pM or lower; (v) 10 pM or lower; (vi) 1 pM
or lower; or (vii) 0.1 pM or lower.
[0174] In some embodiments, the antibody, or antigen-binding
fragment thereof, is administered intravenously. In other
embodiments, the antibody, or antigen-binding fragment thereof, is
administered intramuscularly.
[0175] In some embodiments, the antibody, or antigen-binding
fragment thereof, is administered at a dose of about 100 mg to
about 2000 mg, about 200 mg to about 1500 mg, about 300 mg to about
600 mg, about 500 mg to about 1200 mg, or about 300 mg to about
1200 mg. In some embodiments, the antibody, or antigen-binding
fragment thereof, is administered at a dose of about 200 mg, about
300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg,
about 800 mg, about 900 mg, about 1000 mg about 1100 mg, about 1200
mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg,
about 1700 mg, about 1800 mg, about 1900 mg or about 2000 mg.
[0176] In some embodiments, the antibody, or antigen-binding
fragment thereof, is administered at a dose of about 300 mg
intramuscularly, about 500 mg intravenously, about 600 mg
intramuscularly, or about 1200 mg intravenously.
[0177] In one embodiment, the antibody, or antigen-binding fragment
thereof, is administered once. In one embodiment, the antibody, or
antigen-binding fragment thereof, is administered weekly. In
another embodiment, the antibody, or antigen-binding fragment
thereof, is administered daily, weekly, every two weeks, monthly,
or every two months. In one embodiment, the antibody, or
antigen-binding fragment thereof, is administered weekly for about
four weeks, once weekly for about a month, weekly for about 5
weeks, weekly for about 6 weeks, weekly for about 7 weeks, or
weekly for about two months.
[0178] In one aspect, the present disclosure also relates to kits
comprising: (a) at least one isolated antibody or antigen-binding
antibody fragment disclosed herein; and (b) an instruction for
using the antibody or antigen-binding antibody fragment.
[0179] In some embodiments, the kit may be for use in: (i)
determining whether a CoV is present in a subject; (ii) diagnosing
whether a subject has CoV infection; (iii) predicting whether a CoV
vaccine will elicit a protective immune response; or (iv)
predicting whether a CoV vaccine will elicit a neutralizing
antibody response.
[0180] In one aspect, provided herein are methods of predicting the
in vivo efficacy of an anti-CoV-S antibody or antigen-binding
antibody fragment in preventing or treating CoV infection.
[0181] In some embodiments, the method may comprise: (a) providing
at least one first test subject and at least one second subject or
a cell sample derived from at least one first test subject and at
least one second subject; (b) administering the antibody or
antigen-binding antibody fragment to said at least one first test
subject and said at least one second subject or contacting a cell
sample from said first and second subject with the antibody or
antigen-binding antibody fragment; (c) infecting said at least one
first test subject and said at least one second subject with CoV or
pseudo CoV or a cell sample obtained from said at least one first
test subject and said at least one second subject with CoV or
pseudo CoV; (d) determining whether administration of the antibody
or antigen-binding antibody fragment in (b) results in one or more
of the following compared to a suitable control: (I) reduction in a
CoV-associated symptom; (II) reduction in the CoV viremia; (III)
increase in the survival; (IV) increase in the body weight; or (V)
reduced infection of cells or virus proliferation in cells of the
tested cell sample compared to a control cell sample not contacted
with the antibody or antigen-binding antibody fragment.
[0182] In some embodiments, the method may comprise: (a) providing
at least one first cell sample and at least one second cell sample;
(b) contacting the at least one first cell sample with the antibody
or antigen-binding antibody fragment; (c) infecting said at least
one first cell sample and at least one second cell sample with CoV
or pseudo CoV; (d) determining whether the antibody or
antigen-binding antibody fragment results in one or more of the
following compared to a suitable control: (I) increase in the cell
survival; (II) reduced infection of cells; (III) reduced virus
proliferation; (IV) reduced cell stress or death markers; or (V)
reduced inflammatory cytokines, in cells of the tested cell sample
compared to a control cell sample not contacted with the antibody
or antigen-binding antibody fragment.
[0183] In some embodiments, the method may comprise: (a) providing
at least one first test subject and at least one second subject or
a cell sample derived from at least one first test subject and at
least one second subject; (b) infecting said at least one first
test subject and said at least one second subject with CoV or
pseudo CoV or a cell sample derived from at least one first test
subject and at least one second subject; (c) administering the
antibody or antigen-binding antibody fragment to said at least one
second subject or contacting a cell sample derived from at least
one first test subject and at least one second subject with the
antibody or antigen-binding antibody fragment; (d) determining
whether administration of the antibody or antigen-binding antibody
fragment in (c) results in one or more of the following: (I)
reduction in a CoV-associated symptom; (II) reduction in the CoV
viremia; (III) increase in the survival; (IV) increase in the body
weight; or (V) reduced infection of cells or virus proliferation in
cells in the tested cell sample compared to a control cell sample
not contacted with the antibody or antigen-binding antibody
fragment.
[0184] In some embodiments, the method may comprise: (a) providing
at least one first cell sample and at least one second cell sample;
(b) infecting said at least one first cell sample and at least one
second cell sample with CoV or pseudo CoV; (c) contacting the at
least one first cell sample with the antibody or antigen-binding
antibody fragment; (d) determining whether the antibody or
antigen-binding antibody fragment results in one or more of the
following compared to a suitable control: (I) increase in the cell
survival; (II) reduced infection of cells; (III) reduced virus
proliferation; (IV) reduced cell stress or death markers; or (V)
reduced inflammatory cytokines, in cells of the tested cell sample
compared to a control cell sample not contacted with the antibody
or antigen-binding antibody fragment.
[0185] In one aspect, provided herein are methods of screening for
an antibody or antigen-binding antibody fragment that binds to a
CoV or CoV-S, the method comprising whether an antibody or
antigen-binding antibody fragment comprising 1, 2, 3, 4, 5, or 6
CDRs of any of the antibodies disclosed herein may comprise one or
more of the following features: (i) binds to the S protein of a
CoV; (ii) binds to the S1 subunit of CoV-S; (iii) binds to the RBD
of CoV-S; (iv) binds to the NTD of CoV-S; (v) binds to the
ACE2-binding motif of CoV-S; (vi) competes with ACE2; (vii)
competes with the antibody CR3022; (viii) neutralizes one or more
of SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-229E, HCoV-HKU1, HCoV-NL63,
or HCoV-OK43 or variants thereof; (ix) neutralizes a pseudovirus of
one or more of SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-229E,
HCoV-HKU1, HCoV-NL63, or HCoV-OK43 or variants thereof; (x) results
in reduced infection of cells or virus proliferation in cells in a
susceptible tested cell sample compared to a control cell sample
not contacted with the antibody or antigen-binding antibody
fragment; or (xi) prevents or treats CoV infection in vivo. During
the screening, any of the antibodies disclosed herein and/or an
antibody comprising one or more of the CDRs of the antibodies
disclosed herein may be used as a candidate antibody or a control
antibody.
[0186] In one aspect, the present disclosure also relates to
compositions comprising at least one affinity-matured first
anti-CoV-S antibody or antigen-binding antibody fragment and a
pharmaceutically acceptable carrier or excipient.
[0187] In some embodiments, the at least one first antibody or
antigen-binding antibody fragment may comprise: a VH comprising a
VH CDR1, a VH CDR2, a VH CDR3; and a VL, comprising a VL CDR1 a VL
CDR2, a VL CDR3, and the amino acid sequences of said VH CDR1, said
VH CDR2, said VH CDR3, said VL CDR1, said VL CDR2, and said VL CDR3
are identical to the amino acid sequences of the VH CDR1, VH CDR2,
VH CDR3, VL CDR1, VL CDR2, and VL CDR3, respectively, of an
anti-CoV-S antibody selected from the group consisting of
ADI-58120, ADI-58121, ADI-58122, ADI-58123, ADI-58124, ADI-58125,
ADI-58126, ADI-58127, ADI-58128, ADI-58129, ADI-58130, ADI-58131,
ADI-58130_LCN30cQ, and ADI-59988.
[0188] In particular embodiments, the VH and VL of said first
antibody or antigen-binding antibody fragment may be the VH and VL,
respectively, of an anti-CoV-S antibody selected from the group
consisting of ADI-58120, ADI-58122, ADI-58124, ADI-58126,
ADI-58128, ADI-58130, ADI-58131, and ADI-58130_LCN30cQ.
[0189] In some embodiments, the first antibody or antigen-binding
antibody fragment may comprise an Fc region, optionally wherein the
Fc region may comprise an amino acid sequence of SEQ ID NOs: 11,
12, 13, 14, 15, 16, or 17.
[0190] In one embodiment, the HC and LC of the first antibody or
antigen-binding antibody fragment are the HC and LC, respectively,
of an anti-CoV-S antibody selected from the group consisting of
ADI-58120, ADI-58121, ADI-58122, ADI-58123, ADI-58124, ADI-58125,
ADI-58126, ADI-58127, ADI-58128, ADI-58129, ADI-58130, ADI-58131,
ADI-58130_LCN30cQ, and ADI-59988.
[0191] In certain embodiments, the composition may further comprise
at least one second antibody or antigen-binding antibody fragment
comprising a VH comprising a VH CDR1, a VH CDR2, a VH CDR3 and a
VL, comprising a VL CDR1 a VL CDR2, a VL CDR3. In particular
embodiments, the amino acid sequences of said VH CDR1, said VH
CDR2, said VH CDR3, said VL CDR1, said VL CDR2, and said VL CDR3
may be identical to the amino acid sequences of the VH CDR1, VH
CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, respectively, of an
anti-CoV-S antibody selected from the group consisting of
ADI-58120, ADI-58121, ADI-58122, ADI-58123, ADI-58124, ADI-58125,
ADI-58126, ADI-58127, ADI-58128, ADI-58129, ADI-58130, ADI-58131,
ADI-58130_LCN30cQ, and ADI-59988.
[0192] In some embodiments, the second antibody or antigen-binding
antibody fragment may comprise an Fc region, optionally wherein the
Fc region may comprise an amino acid sequence of SEQ ID NOs: 11,
12, 13, 14, 15, 16, or 17.
[0193] In particular embodiments, the antibody or antigen-binding
antibody fragment according to the present disclosure may
comprise:
(217) a HC comprising the amino acid sequence of SEQ ID NO: 21701
and a LC comprising the amino acid sequence of SEQ ID NO: 21711;
(218) a HC comprising the amino acid sequence of SEQ ID NO: 21801
and a LC comprising the amino acid sequence of SEQ ID NO: 21811;
(219) a HC comprising the amino acid sequence of SEQ ID NO: 21901
and a LC comprising the amino acid sequence of SEQ ID NO: 21911;
(220) a HC comprising the amino acid sequence of SEQ ID NO: 22001
and a LC comprising the amino acid sequence of SEQ ID NO: 22011;
(221) a HC comprising the amino acid sequence of SEQ ID NO: 22101
and a LC comprising the amino acid sequence of SEQ ID NO: 22111;
(222) a HC comprising the amino acid sequence of SEQ ID NO: 22201
and a LC comprising the amino acid sequence of SEQ ID NO: 22211;
(223) a HC comprising the amino acid sequence of SEQ ID NO: 22301
and a LC comprising the amino acid sequence of SEQ ID NO: 22311;
(224) a HC comprising the amino acid sequence of SEQ ID NO: 22401
and a LC comprising the amino acid sequence of SEQ ID NO: 22411;
(225) a HC comprising the amino acid sequence of SEQ ID NO: 22501
and a LC comprising the amino acid sequence of SEQ ID NO: 22511;
(226) a HC comprising the amino acid sequence of SEQ ID NO: 22601
and a LC comprising the amino acid sequence of SEQ ID NO: 22611;
(227) a HC comprising the amino acid sequence of SEQ ID NO: 22701
and a LC comprising the amino acid sequence of SEQ ID NO: 22711;
(228) a HC comprising the amino acid sequence of SEQ ID NO: 22801
and a LC comprising the amino acid sequence of SEQ ID NO: 22811;
(229) a HC comprising the amino acid sequence of SEQ ID NO: 22901
and a LC comprising the amino acid sequence of SEQ ID NO: 22911;
(230) a HC comprising the amino acid sequence of SEQ ID NO: 23001
and a LC comprising the amino acid sequence of SEQ ID NO: 23011; or
(231) a HC comprising the amino acid sequence of SEQ ID NO: 23101
and a LC comprising the amino acid sequence of SEQ ID NO:
23111.
[0194] In further embodiments, the composition according to the
present disclosure may comprise: (A) at least one first antibody or
antigen-binding antibody fragment selected from the group
consisting of the antibodies or antigen-binding antibody fragments
comprising the HC and LC combination of (217)-(231) as described
above; and (B) a pharmaceutically acceptable carrier or
excipient.
[0195] In yet further embodiments, the composition may additionally
comprise at least one second antibody or antigen-binding antibody
fragment selected from the group consisting of the antibodies or
antigen-binding antibody fragments comprising the HC and LC
combination of (217)-(231) as described above.
[0196] Additionally, the present disclosure further encompasses
isolated antibodies and antigen-binding antibody fragments thereof,
which competes for binding with any one or more of the anti-CoV
antibodies or antigen-binding antibody fragments thereof as
described herein.
[0197] The present disclosure also encompasses isolated antibodies
or antigen-binding antibody fragments thereof, which bind the same
epitope as any one or more of the anti-CoV antibodies or
antigen-binding antibody fragments thereof as described herein.
[0198] The present disclosure further encompasses affinity matured
variants of any one or more of the anti-CoV antibodies or
antigen-binding antibody fragments thereof as described herein.
[0199] In one aspect, disclosed herein is a method of treating a
coronavirus infection by SARS-CoV, SARS-CoV-2, and/or another
coronavirus optionally selected from the group consisting of
MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 in a
subject in need thereof, the method comprising administering to the
subject a therapeutically effective amount of an antibody, or
antigen-binding antibody fragment thereof, which binds the same
epitope as ADI-58125, and/or which competes for binding with
ADI-58125.
[0200] In one aspect, disclosed herein is a method of decreasing
the risk of mortality, hospitalization, mechanical ventilation, or
a combination thereof in a patient infected by SARS-CoV, ARS-CoV-2,
and/or another coronavirus optionally selected from the group
consisting of MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-229E, and
HCoV-NL63, the method comprising administering to the subject a
therapeutically effective amount of an isolated antibody, or
antigen-binding antibody fragment thereof, which binds the same
epitope as ADI-58125, and/or which competes for binding with
ADI-58125.
[0201] In another aspect, disclosed herein is a method of
preventing infection of a subject by SARS-CoV, SARS-CoV-2, and/or
another coronavirus optionally selected from the group consisting
of MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63, the
method comprising administering to the subject a therapeutically
effective amount of an isolated antibody, or antigen-binding
antibody fragment thereof, which binds the same epitope as
ADI-58125, and/or which competes for binding with ADI-58125.
[0202] In one embodiment, the antibody, or antigen-binding fragment
thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55688. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55689. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55690. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55691. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55692. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55693. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55694. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55695. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55696. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55697. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55698. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55699. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55700. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55701. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55702. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55703. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55704. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55705. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55706. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55707. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55708. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55709. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55710. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55711. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55712. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55713. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55714. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55715. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55716. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55717. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55718. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55719. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55721. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55722. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55723. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55724. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55725. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55726. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55727. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55728. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55729. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55730. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55731. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55732. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55733. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55734. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55735. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55736. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55737. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55738. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55739. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55740. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55741. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55742. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55743. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55744. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55745. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55746. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55747. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55748. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55749. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55750. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55751. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55752. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55753. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55754. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55755. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55756. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55757. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55758. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55720. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55760. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55761. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55762. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55763. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55765. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55766. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55767. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55769. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55770. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55771. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55775. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55776. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55777. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55950. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55951. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55952. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55953. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55954. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55955. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55956. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55957. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55958. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55959. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55960. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55961. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55962. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55963. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55964. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55965. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55966. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55967. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55968. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55969. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55970. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55972. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55973. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55974. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55975. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55976. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55977. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55978. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55979. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55980. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55981. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55982. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55984. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55986. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55988. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55989. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55990. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55992. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55993. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55994. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55995. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-55996. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-55997. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-55998. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-55999. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56000. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56001. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56002. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1
, FIG. 2 or FIG. 36 is ADI-56003. In one embodiment, the antibody,
or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36
is ADI-56004. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56005
ADI-56006. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56007. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56008. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56009. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56010. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56011. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56012. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56013. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56014. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56015. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56016. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56017. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56018. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56019. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56020. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56021. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56022. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56023. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56024. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56025. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56026. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56027. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56028. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56029. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56030. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56031. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56032. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56033. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56034. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56035. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56037. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56038. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56039. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56040. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56041. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56042. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56043. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56044. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56045. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56046. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56047. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56048. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56049. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56050. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56051. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56052. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56053. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56054. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56055. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56056. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56057. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56058. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56059. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56061. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56062. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56063. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56064. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56065. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56066. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56067. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56068. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56069. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56070. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56071. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56072. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56073. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56074. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56075 ADI-56076. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56078. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56079. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56080. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56081. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56082. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-56083. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-56084. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-56443. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-56479. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-58120. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-58121. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-58122. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-58123. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-58124. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-58125. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-58126. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-58127. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is ADI-58128. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
FIG. 1, FIG. 2 or FIG. 36 is ADI-58129. In one embodiment, the
antibody, or antigen-binding fragment thereof, of FIG. 1, FIG. 2 or
FIG. 36 is ADI-58130. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-58131. In one embodiment, the antibody, or antigen-binding
fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-58130_LCN30cQ. In one embodiment, the antibody, or
antigen-binding fragment thereof, of FIG. 1, FIG. 2 or FIG. 36 is
ADI-59988.
BRIEF DESCRIPTION OF THE DRAWINGS
[0203] FIGS. 1A-1I provide the SEQ ID NOs assigned to the
respective VH FR1, VH CDR1, VH FR2, VH CDR2, VH FR3, VH CDR3, and
VH FR4 amino acid sequences, the respective VH amino acid
sequences, and the respective VH-encoding nucleic acid sequences
for 211 antibodies as indicated.
[0204] FIGS. 2A-2I provide the SEQ ID NOs assigned to the
respective VL FR1, VL CDR1, VL FR2, VL CDR2, VL FR3, VL CDR3, and
VL FR4 amino acid sequences, the respective VL amino acid
sequences, and the respective VH-encoding nucleic acid sequences
for 211 antibodies as indicated.
[0205] FIGS. 3A-3E provide the germline origin of the VH and VL of
each of the 211 antibodies.
[0206] FIGS. 4A-4I provide the number of nucleotide substitutions
in the VH-encoding and VL-encoding sequences as compared to the
germline sequences and the number of amino acid alterations in the
VH and VL polypeptide sequences as compared to the germline-encoded
VH and VL sequences, for each of the antibodies. Eight antibodies
were not analyzed (in this and some of other studies) due to the
high sequence similarity to another antibody within the 211
antibodies, and were shown as blank.
[0207] FIGS. 5A-5C provide the B cell isotype of the 211
antibodies.
[0208] FIGS. 6A-6E provide the summary of SARS-CoV-2-S-binding B
cell isolation and determination of the germline origin and isotype
of each of the isolated antibodies. FIG. 6A shows the frequency of
SARS-CoV-2 S-reactive B cells in VRC #202367 (a convalescent
SARS-CoV donor) and a negative control SARS-CoV naive donor.
Fluorescence activated cell sorting (FACS) plots shown are gated on
CD19*CD20*IgDIgM B cells. SARS-CoV-2 S was labeled with two
different colors to reduce background binding. FIG. 6B shows
binding of 315 isolated antibodies to SARS-CoV-2 S, as determined
by BLI. The solid line indicates the threshold used for designating
binders (0.1 RUs). FIG. 6C shows clonal lineage analysis. Each
lineage is represented as a segment proportional to the lineage
size. Antibodies that utilize VH1-69/VK2-30 germline gene pairing
are shown in blue. Other germlines are shown in dark gray. The
total number of isolated antibodies is shown in the center of the
pie. Clonal lineages were defined based on the following criteria:
identical VH and VL germline gene, identical VH CDR3 length, and VH
CDR3 amino acid identity .gtoreq.80%.
[0209] FIG. 6D shows the load of somatic mutations, expressed as
number of nucleotide substitutions in VH, in unique antibodies and
members of expanded clonal lineages. FIG. 6E shows the percentage
of antibodies having the IgA+ isotype and antibodies having the
IgG+ isotype. Proportion of SARS-CoV-2-S-binding antibodies derived
from IgG.sup.+ and IgA.sup.+ B cells, as determined by index
sorting. Statistical comparisons were made using the Mann-Whitney
test (****P<0.0001). Red bars indicate medians. RU, response
unit; VH, variable region of the heavy chain.
[0210] FIGS. 7A-7I show part of the results from the binding assays
in Example 2. The KD [M], k.sub.on [M.sup.-1 s.sup.-1], k.sub.off
[s.sup.-1], and Binding Response (nm) for the S protein of SARS-CoV
and SARS-CoV-2, for the 211 antibodies, are provided.
[0211] FIGS. 8A-8E show part of the results from the binding assays
in Example 3. The KD [M], k.sub.on [M.sup.-1 s.sup.-1], k.sub.off
[s.sup.-1], and Binding Response (n) for the S protein of HCoV-229E
(an endemic/seasonal/circulating species), for the 211 antibodies,
are provided.
[0212] FIGS. 9A-9E show part of the results from the binding assays
in Example 3. The KD [M], k.sub.on [M.sup.-1 s.sup.-1], k.sub.off
[s.sup.-1], and Binding Response (nm) for the S protein of
HCoV-HKU1 (an endemic/seasonal/circulating species), for the 211
antibodies, are provided.
[0213] FIGS. 10A-10E show part of the results from the binding
assays in Example 3. The KD [M], k.sub.on [M.sup.-1 s.sup.-1],
k.sub.off [s.sup.-1], and Binding Response (nm) for the S protein
of HCoV-NL63 (an endemic/seasonal/circulating species), for the 211
antibodies, are provided.
[0214] FIGS. 11A-11E show part of the results from the binding
assays in Example 3. The KD [M], k.sub.on [M.sup.-1 s.sup.-1],
k.sub.off [s.sup.-1], and Binding Response (nm) for the S protein
of HCoV-OC43 (an endemic/seasonal/circulating species), for the 211
antibodies, are provided.
[0215] FIGS. 12A-12I provide a summary of the binding affinity
results from FIGS. 7-11. When an antibody showed a binding response
(nm) value of 0.1 or higher for a target, the antibody was
considered as a binder (shown as "Yes") to that target. Note that
antibodies with a binding response value such as 0.099 or 0.098
that can be round to 0.1 were also considered as binders and shown
as "Yes". When an antibody showed a binding response (nm) value of
<0.1 for a target in FIGS. 7-11, the antibody was considered as
a non-binder (shown as "No") for that target. When an antibody was
"Yes" for SARS-CoV-S and/or SARS-CoV-2-S but "No" for all of S
protein of the circulating CoV species (HCoV-229E, HCoV-HKU,
HCoV-NL63, and HCoV-OK43), the antibody is classified as "SARS1-2
specific". When an antibody was "Yes" for SARS-CoV-S and/or
SARS-CoV-2-S and also "Yes" for the S protein of one or more the
circulating CoV species (HCoV-229E, HCoV-HKU, HCoV-NL63, and
HCoV-OK43), the antibody is classified as "Broad".
[0216] FIGS. 13A-13C provide the polyspecificity score and
polyspecificity category for each antibody.
[0217] FIG. 13D provides polyspecificity of broadly cross-reactive
("Broad") and SARS-CoV/SARS-CoV-2-specific ("SARS1/2 only")
antibodies, as determined using a previously described assay (Xu et
al., Protein Engineering, Design and Selection 26(10), 663-670
(2013)). Thresholds for high, medium, low, and no polyspecificity
are indicated by dashed lines. Polyspecificity scores for 138
clinical antibodies are shown for comparison (Jain et al., PNAS
1114(5), 944-949 (2017).
[0218] FIGS. 14A-14L contain a summary of the binding properties of
SARS-CoV-2 S-specific antibodies based on the results from Examples
2 and 3. FIG. 14A shows apparent binding affinities of SARS-CoV-2
S-specific IgGs to recombinant prefusion-stabilized SARS-CoV-S and
SARS-CoV-2-S proteins, as determined by BLI measurements.
Antibodies for which binding curves could not be fit are designated
as "p.f" (meaning "poor fit") on the plot. The majority of tested
mAbs (153 out of 202) binds to both SARS-CoV-2-S and SARS-CoV-S,
while a subset of mAbs are SARS-CoV-2 S-specific and that over half
of the cross-reactive antibodies (89/153) bound with KDApp>10 nM
to both SARS-CoV and SARS-CoV-2 S. FIG. 14B provides a heat map
showing K.sub.D.sup.Apps of the isolated antibodies for SARS-CoV,
SARS-CoV-2, 229E, HKU1, NL63, and OC43 S proteins. Germline gene
usage, clonal expansion, and SHM are indicated in the three
leftmost panels. N.B., non-binder. FIG. 14C provides the load of
somatic mutations in broadly cross-reactive ("Broad") and
SARS-CoV/SARS-CoV-2-specific ("SARS1/2 only") antibodies. Black
bars indicate medians. FIG. 14D provides the degree of clonal
expansion in broadly cross-reactive ("Broad") and
SARS-CoV/SARS-CoV-2-specific ("SARS1/2 only") antibodies. Each
lineage is represented as a segment proportional to the lineage
size. The total number of antibodies is shown in the center of the
pie. FIG. 14E shows the proportion of broadly cross-reactive
("Broad") and SARS-CoV/SARS-CoV-2-specific ("SARS1/2 only")
antibodies derived from IgG.sup.+ and IgA.sup.+ B cells, as
determined by index sorting. FIG. 14F shows the load of somatic
mutations in SARS-CoV-2 S-reactive antibodies isolated from three
naive donors and VRC #202367. Antibodies from naive donors are
pooled for this analysis. FIG. 14G compares the binding activity of
SARS-CoV-2 S-reactive antibodies isolated from three naive donors
and VRC #202367, as determined by BLI. The statistical significance
(**) is between the data from VRC #202367 and pooled data from
three naive donors FIG. 14H shows VH/VL germline gene usage of
SARS-CoV-2 S-specific antibodies that display cross-reactivity with
circulating HCoV S proteins.
[0219] FIGS. 141 and 14J show ELISA binding reactivity of healthy
donor sera to SARS-CoV-2, SARS-CoV, and circulating HCoV S
proteins. CR3022 is included as a positive control. FIG. 14K shows
the percentage of SARS-CoV-2 S-specific B cells observed in each
naive donor, as determined by flow cytometry. FIG. 14L shows the
clonal lineage analysis. Each lineage is represented as a segment
proportional to the lineage size. The total number of isolated
antibodies is shown in the center of each pie. Clonal lineages were
defined based on the following criteria: identical VH and VL
germline gene, identical CDR H3 length, and CDR H3 amino acid
identity >80%. Clonal lineages are not shown for Donor 1 because
only three SARS-CoV-2 S-specific antibodies were isolated from this
donor.
[0220] FIGS. 15A-15I show part of the results from the epitope
mapping assays in Example 4. The K.sub.D [M], k.sub.on [M.sup.-1
s.sup.-1], k.sub.off [s.sup.-1], and Binding Response (nm) data for
the S1 subunit (monovalent or bivalent ("AVID")) of the S protein
of SARS-CoV-2, for each antibody are provided.
[0221] FIGS. 16A-16E show part of the results from the epitope
mapping assays in Example 4. The K.sub.D [M], k.sub.on [M.sup.-1
s.sup.-1], k.sub.off [s.sup.-1], and Binding Response (nm) data for
the S2 subunit of the S protein of SARS-CoV-2, for each antibody
are provided.
[0222] FIGS. 17A-17I show part of the results from the epitope
mapping assays in Example 4. The K.sub.D [M], k.sub.on [M.sup.-1
s.sup.-1], k.sub.off [s.sup.-1], and Binding Response (nm) data for
the RBD domain (monovalent or bivalent ("AVID") of the S protein of
SARS-CoV-2, for each antibody are provided.
[0223] FIGS. 18A-18E show some of the results from the epitope
mapping assays in Example 4. The K.sub.D [M], k.sub.on [M.sup.-1
s.sup.-1], k.sub.off [s.sup.-1], and Binding Response (n) data for
the N-terminal domain (NTD) of the S protein of SARS-CoV-2, for
each antibody are provided.
[0224] FIGS. 19A-19I provide a summary of the binding affinity
results from FIGS. 15-18. In these figures, when an antibody showed
a binding response (n) value of 0.1 or higher for a target, the
antibody was considered as a binder (shown as "Yes") to that
target. Note that for antibodies with a binding response value such
as 0.099 or 0.098 that can be round up to 0.1; these antibodies
were also identified as binders and shown as "Yes". By contrast,
when an antibody showed a binding response (nm) value of <0.1
for a target in FIGS. 15-18, the antibody was considered as a
non-binder (shown as "No") for that target.
[0225] FIGS. 20A-20F provide the results from the cell binding
assays in Example 5. FIGS. 20A-20E show binding of each antibody to
cells engineered to express the S protein of SARS-CoV-2 on the
surface is shown in fold binding over background and in EC50 [nM].
FIG. 20F shows binding of SARS-CoV-2 S-specific antibodies to
SARS-CoV S expressed on the surface of cells. As shown therein none
of the antibodies tested showed significant binding to recombinant
prefusion-stabilized SARS-CoV S, as determined by BLI. In these
binding assays antibodies were tested at a 100 nM
concentration.
[0226] FIGS. 21A-21E show the results from the binding competition
assays in Example 4. The figures show whether the binding of each
tested antibody competed with ACE2 or a commercially available
antibody, CR3022.
[0227] FIG. 21F is a summary of results shown in FIGS. 15-18 and
21. The chart provides the proportion of different antigenic sites
within SARS-CoV-2-S of the 65 tested antibodies that bind to
SARS-CoV-2-S with K.sub.D.sup.Apps<10 nM. The different
antigenic sites are the S2 subunit, the NTD within the S1 subunit,
and the RBD within the S1 subunit. As shown therein of the
antibodies that bound to the RBD, only some competed with hACE2.
Those which did not compete with hACE2 are also shown.
[0228] FIGS. 22A-22I show the results from the neutralization
assays in Example 6.
[0229] FIGS. 23A-23E contain analyses of the relationship between
the binding epitope and the neutralizability of the antibodies.
FIG. 23A provides a graph showing percent authentic SARS-CoV-2
infection observed in the presence of each antibody identified by
"Antibody index" (x axis) at a concentration of 100 nM. The
corresponding antibody names are shown in Table 5 below. Bars are
colored by targeted antigenic site. FIG. 23B provides a graph
showing the % neutralization of authentic SARS-CoV-2 virus of each
antibody (y axis) by the epitope/antigenic site within SARS-CoV-2-S
(x axis). For RBD-binding antibodies, the target is further
specified by whether the antibody competes with hACE2. FIG. 23C
provides a heat map showing the neutralizing activities and
competitive binding profiles of the RBD-directed antibodies. FIG.
23D provides neutralization of authentic SARS-CoV-2 (strain
n-CoV/USA_WA1/2020) by antibodies as measured by a
micro-neutralization assay on Vero E6 cells. The negative control
antibody, ADI-26140, is specific for chicken egg lysozyme. CR3022
is included for comparison. FIG. 23E provides a dot plot showing
binding to cell-surface SARS-CoV-2 S (shown in FOB on y axis)
versus SARS-CoV-2 neutralization at a 100 nM concentration (shown
in % on x axis). Each point represents a single antibody and data
points are colored by the antigenic site. In the figure "FOB"
refers to fold over background.
[0230] FIGS. 23F-23G show the results from the neutralization
assays on selected antibodies in Example 6. FIG. 23F provides
neutralization of SARS-CoV and SARS-CoV-2 MLV pseudovirus (strain
n-CoV/USA_WA1/2020) by eight antibodies using HeLa-ACE2 target
cells. FIG. 23G provides neutralization of authentic SARS-CoV and
SARS-CoV-2 using Vero E6 target cells. Data represents two
technical replicates. Antibodies with black arrow were selected for
affinity maturation in Example 12.
[0231] FIG. 24 provides graphs showing correlation between
authentic SARS-CoV-2 neutralization IC50 using Vero E6 target cells
and (top) MLV-SARS-CoV-2 pseudovirus neutralization IC50 using
HeLa-ACE2 target cells or (bottom) VSV-SARS-CoV-2 neutralization
IC50 using Vero E6 target cells. R2 values were calculated using
linear regression.
[0232] FIG. 25 provides a summary of neutralization IC50 [ug/mL]
values for neutralization assays using nine selected antibodies and
different viruses as shown therein.
[0233] FIG. 26 provides a summary of authentic SARS-CoV-2
neutralization IC50 [ug/mL] values and of affinities (K.sub.D
values) to SARS-CoV-S and SARS-CoV-2-S, for seven selected
antibodies. Based on their binding properties some of these
antibodies were selected for further affinity maturation.
[0234] FIGS. 27A-27C provide clusters based on the VH CDR3
sequences among selected antibodies, as tested in Example 7.
[0235] FIGS. 28A-28B provide sample plots for the clusters
containing more than 2 antibodies, i.e., Clusters 1 through 5.
[0236] FIG. 29 provides the amino acid changes introduced by
affinity maturation in Example 12. Amino acid changes for
ADI-57983, ADI-57978, and ADI-56868 are relative to the sequence
the parent (i.e., before affinity maturation in Example 12)
antibody, ADI-55689, ADI-55688, and ADI-56046, respectively.
[0237] FIG. 30A provides a graph comparing affinity to SARS-CoV-2-S
of parent antibodies (i.e., before affinity maturation in Example
12) and affinity maturation progenies (after one or two cycles),
measured in Example 13.
[0238] FIG. 30B provides a graph comparing parent antibodies (i.e.,
before affinity maturation in Example 12) and affinity maturation
progenies for the ability to neutralize authentic SARS-CoV-S and
SARS-CoV-2, measured in Example 14. Data points corresponding to
the lead progeny derived from ADI-55689 are marked "ADI-57983".
[0239] FIG. 30C provides representative yeast surface display
library selections. Four flow cytometry panels on the left show
flow cytometric sorting of yeast display libraries containing
diversity in the ADI-55688 HC (top) or LC (bottom). Libraries were
sorted for improved binding to the SARS-CoV-2 S1 protein relative
to the parent clone. Round 1 gates indicate the yeast populations
that were sorted for a second round of selection, and round 2 gates
indicate the yeast populations that were sorted for amplification
of heavy- or light-chain variable region genes and transformation
into yeast for HC/LC combinatorial library generation. Two flow
cytometry panels on the right shown flow cytometric sorting of
HC/LC combinatorial libraries. Libraries were sorted for improved
binding to the SARS-CoV-2 S1 protein relative to the round 2 output
of the HC diversity libraries. The round 1 gate indicates the yeast
population that was sorted for a second round of selection and the
round 2 gate indicates the yeast population that was sorted for
individual colony picking. The scheme illustrates the combined
heavy chain and light chain selection for affinity maturation in
Example 12,
[0240] FIG. 30D provides flow cytometry plots from the terminal
round of selection showing binding of parental antibodies and
affinity maturation libraries to the SARS-CoV-2 S1 protein at a 1
nM concentration. Gates indicate the yeast populations sorted for
antibody sequencing and characterization.
[0241] FIG. 31A provides summary information regarding the origin
(donor, B cell phenotype, and, if applicable, lineage (i.e., the
antibody before affinity maturation induced in Example 12)).
epitope within the S protein of SARS-CoV-2, and neutralization IC50
values for five selected antibodies.
[0242] FIG. 31B provides a summary of antibody developability
parameters measured in Example 18 for five selected antibodies.
"pI", isoelectric point; "PSR", interaction with polyspecificity
reagent; "HIC", Hydrophobic interaction chromatography; "Tm",
melting temperature.
[0243] FIGS. 32A-32H provide the results from Examples 16 and 19,
the binding kinetics of two antibodies isolated from convalescent
COVID-19 patients in Example 15. The binding specificity summary
(FIG. 32A) and individual binding parameters for SARS-CoV-2-S (FIG.
32B), for the RBD of SARS-CoV-2-S (FIG. 32C), for the NTD of
SARS-CoV-2-S (FIG. 32D), for S1 subunit of SARS-CoV-2-S (FIG. 32E),
for S2 subunit of SARS-CoV-2-S (FIG. 32F), and for the seasonal CoV
HKU1 S protein (FIG. 32G), and competition assay results (FIG. 32H)
are provided.
[0244] FIGS. 32I-32J provide the results from the cross-competition
study in Example 20 for selected antibodies.
[0245] FIG. 33 provide the results from neutralization assays in
Example 17 on two antibodies isolated from convalescent COVID-19
patients.
[0246] FIG. 34 provides the amino acid changes made in the five
antibodies (ADI-58120, ADI-58124, ADI-58126, ADI-58128, and
ADI-58130) to fix the mutation(s) away from the germline-encoded
sequence caused by the degenerate primers to match the
germline-encoded sequence.
[0247] FIG. 35 provides the germline origin of the VH and VL and
the number of amino acid or nucleotide substitutions relative to
the germline-encoded or greyline sequence, respectively, in the VH
and VL of ADI-58120, ADI-58124, ADI-58126, ADI-58128, and ADI-58130
(antibodies after the fixation of "primer mutation" back to the
germline sequence as described in FIG. 34).
[0248] FIG. 36A provides the SEQ ID NOs assigned to the respective
VH FR1, VH CDR1, VH FR2, VH CDR2, VH FR3, VH CDR3, and VH FR4 amino
acid sequences, the respective VH amino acid sequences, the
respective heavy chain amino acid sequences, and the respective
VH-endcoding DNA sequences for different antibodies.
[0249] FIG. 36B provides the SEQ ID NOs assigned to the respective
VL FR1, VL CDR1, VL FR2, VL CDR2, VL FR3, VL CDR3, and VL FR4 amino
acid sequences, the respective VL amino acid sequences, and the
respective light chain amino acid sequences, and the respective
VL-endcoding DNA sequences for different antibodies.
[0250] FIG. 37A provides exemplary binding kinetics of pre-affinity
maturation antibodies (ADI-55689, ADI-55688, and ADI-56046) and
post-affinity maturation antibodies derived therefrom,
respectively, after fixing primer mutations (ADI-58120, ADI-58124,
and ADI-58126). The SPR sensorgrams show binding of each Fab to
SARS-CoV-2 RBD-SD1. Binding data are shown as black lines and the
best fit of a 1:1 binding model is shown as red lines.
[0251] FIG. 37B provides a comparison of the affinity to SARS-CoV-2
S protein of ADI-58124 and its parental antibodies, which are
antibodies used to obtain ADI-58124 by affinity maturation (i.e.,
ADI-55688) and cycle 1 and cycle 2 affinity maturation
progenies.
[0252] FIG. 37C provides a table summarizing binding kinetics of
ADI-58125 as a full IgG or a Fab to SARS-CoV-2 S protein,
SARS-CoV-2 RBD, SARS-CoV S protein, SARS-CoV RBD, or WIV-1 RBD.
[0253] FIGS. 38A-38D compare biophysical properties of antibodies
according to the present disclosure, anti-SARS-CoV-2 antibodies
currently under clinical trials, and 42 clinically approved
antibodies (Jain T. et al., Proc Natl Acad Sci USA. 2017 Jan. 31;
114(5):944-949). FIG. 38A compares polyreactivity scores, as
determined as described previously (L. Shehata et al., Cell Reports
28, 3300-3308 e3304 (2019)). The thresholds for high, low, and
clean polyreactivity were defined based on a previously reported
correlation between polyreactivity score and serum half-life in
humans (L. Shehata et al., Cell Reports 28, 3300-3308 e3304
(2019)). FIG. 38B compares hydrophobicity, as determined by
hydrophobic interaction chromatography. FIG. 38C compares
self-interaction propensity (left), as determined by
affinity-capture self-interaction nanoparticle spectroscopy (Liu Y.
et al., MAbs. March-April 2014; 6(2):483-92.). FIG. 38D compares
thermal stability (right) defined by Fab melting temperatures, as
determined by differential scanning fluorimetry (DSF).
[0254] FIGS. 39A-39F compares neutralization of different
coronaviruses and pseudoviruses by ADI-58124 and its parent
ADI-55688, and anti-SARS-CoV-2 antibodies currently under clinical
trials. FIG. 39A provides a dot plot showing MLV-SARS-CoV-2
pseudovirus neutralization IC50s of parental antibodies and
affinity matured progenies. FIG. 39B provides a heat map (top) and
a graph (bottom) showing the neutralization IC50s of the indicated
antibodies against authentic SARS-CoV, WIV-1-nluc, SHC014-nluc,
SARS-CoV-2-nluc, and SARS-CoV-2 using either HeLa-ACE2 or Vero
target cells. SARS-CoV, WIV-1-nluc, SHCO14-nluc, and SARS-CoV-2
nluc assays were run using Vero target cells. WIV-1-nluc,
SHC014-nluc, and SARS-CoV-2-nluc are recombinant,
reverse-genetics-derived viruses encoding a nano-luciferase
reporter gene. FIGS. 39C and 39D provide individual neutralization
curves of pre- and post-affinity maturation antibodies and clinical
antibodies against authentic SARS-CoV, WIV1-nLuc, SHCO14-nLuc, and
mouse adapted SARS-CoV-2-MA2-nLuc ("SARS2-nLuc") viruses on Vero E6
cells. FIG. 39E provides authentic SARS-CoV-2 neutralization
titrations with ADI-58124, performed using either HeLa-ACE2 (left)
or Vero (right) target cells. The curves were fitted by nonlinear
regression (log[inhibitor] vs. normalized response, variable
slope). IC50 values for ADI-58125 and ADI-58129, along with
clinical antibodies are also provided. FIG. 39F shows exemplary
neutralizing activity by ADI-58124 against WA1 strain of
SARS-CoV-2, WT or D614G, measured using an MLV-based pseudovirus
assay in HeLa-ACE2 target cells.
[0255] FIGS. 39G-39M provide exemplary neutralization data obtained
with antibodies according to the present disclosure having a
wild-type or non-wild type Fc and with anti-SARS CoV-2 antibodies
under clinical trials. FIG. 39G shows neutralization of authentic
SARS-CoV-2 by ADI-58125 assayed using ELISA in HeLa-ACE2 cells
(top) and VeroE6 cells (bottom). FIG. 39H shows neutralization of
SHC014 (top left) and WIV1 (top right) SARS-like coronaviruses from
bats (both encoding luciferase reporter) by ADI-58125 assayed using
the luciferase assay system in VeroE6 cells. IC50 and IC90 values
for ADI-58125 and IC50 values for other antibodies are also
provided.
[0256] FIG. 39I shows neutralization of SARS-CoV pseudotype MLV
virus (left), SARS-CoV-2 pseudotype MLV virus (center), or
SARS-CoV-2 D614G pseudotype MLV virus (right) (all encoding
luciferase reporter) by ADI-58125 assayed using the luciferase
assay system in HeLa-ACE2 cells. IC50 and IC90 values for ADI-58125
and IC50 values for ADI-58129 and clinical antibodies are also
provided.
[0257] FIG. 39J shows exemplary inhibition of authentic SARS-CoV
infection by indicated antibodies assayed using the method
described herein in Vero E6 cells. IC50 values are also provided.
FIGS. 39K-39M provide exemplary neutralizing activity by indicated
antibodies against SARS-CoV pseudotype MLV virus (top left),
SARS-CoV-2 pseudotype MLV virus (top right), and SARS CoV-2 D614G
mutant pseudotype MLV virus (bottom), respectively, assayed using
the luciferase assay system in HeLa-ACE2 cells.
[0258] FIG. 39N provides authentic SARS-CoV-2 neutralization
titrations with ADI-58125 performed using either HeLa-ACE2 (top) or
Vero (bottom) target cells. The curves were fitted by nonlinear
regression (log[inhibitor] vs. normalized response, variable
slope).
[0259] FIG. 39O provides a comparison of IC50 values (top) and
neutralization plateau (bottom) for ADI-58125 and ADI-58129, along
with clinical antibodies.
[0260] FIGS. 40A-40J compares the breadth of binding to diverse
sarbecoviruses and circulating SARS-CoV-2 variants by ADI-58120,
ADI-58124, ADI-58125, and ADI-58126, and anti-SARS-CoV-2 antibodies
in clinical trials. ADI-58125 and ADI-58124 share the same CDR
sequences and only differ in the Fc region which has been
engineered for half-life extension purpose. FIG. 40A provides a
phylogenetic tree of 57 sarbecoviruses constructed from mafft and
maximum likelihood analysis of RBD-SD1 amino acid sequences
extracted from the European Nucleotide Archive and GISAID database.
Representative sarbecovirus RBDs included for further study are in
bold text and colored according to their canonical phylogenetic
lineages. FIG. 40B provides a heat map of apparent equilibrium
dissociation constants (K.sub.D.sup.App) against 17 representative
yeast-displayed RBDs, determined by normalized nonlinear regression
fitting. FIG. 40C provides binding to yeast-displayed RBD of
naturally-occurring SARS-CoV-2 variants. Briefly, SARS-CoV-2
sequences were retrieved from the GISAID database on Jul. 14, 2020
(n=63551). Mutants observed more than 6 times and published
antibody escape mutants also observed in the database were chosen
for analysis. Binding signal was normalized to RBD expression and
calculated as percent antibody binding to the variant SARS-CoV-2
RBD relative to the WT SARS-CoV-2 RBD, assessed at their respective
K.sub.D.sup.Appconcentrations for the WT RBD construct. The
prevalence of each variant, calculated from deposited sequences on
Oct. 19, 2020 (n=148115), is shown as a percentage of the total
number of sequences analyzed. FIG. 40D provides a graph showing
association between the number of natural SARS-CoV-2 variants with
observed loss of binding, defined as less than 25% of WT SARS-CoV-2
binding, and percentage of Clade I sarbecovirus RBDs recognized by
individual antibodies. FIG. 40E provides a heat map of apparent
equilibrium dissociation constants (K.sub.D.sup.App) against 17
representative yeast-displayed RBDs, determined by normalized
nonlinear regression fitting. KD (nM) values for ADI-58120,
ADI-58125, ADI-58126, and clinical antibodies are also provided.
FIG. 40F provides binding of ADI-58124 to yeast-displayed RBD of
naturally occurring SARS-CoV-2 variants. FIG. 40G provides a
comparison between binding of ADI-58125 to yeast-displayed RBD of
naturally occurring SARS-CoV-2 variants with other clinical
antibodies. FIG. 40H depicts that ADI-58125 binds with comparable
affinity to all common circulating SARS-CoV-2 variants and emerging
lineages B.1.1.7/501Y.V1 (UK), B.1.351/501Y.V2 (South African) and
P.1/501Y.V3 (Brazilian). FIG. 40I depicts that neutralization of
P.1., Victoria, B.1.17 and B.1351 strains by a panel of human
monoclonal antibodies. ADI-58122, ADI-58125 and ADI-58127 retained
high affinity binding to P.1 variant with all reaching a plateau at
100% neutralization.
[0261] FIG. 40J depicts antibody mediated inhibition of SARS-CoV-2
spike (S) protein binding to human ACE2. S binding to ACE2-coated
plates was assessed in the presence of varying concentration of
selected antibodies in an ELISA assay.
[0262] FIGS. 41A-41G demonstrate that ADI-58120, ADI-58124,
ADI-58126, and ADI-58128 respectively bind to a conserved epitope
on the SARS-CoV-2 RBD overlapping with the hACE2 binding site. FIG.
41A provides a schematic showing the generation and selection of a
mutagenized, yeast surface-displayed SARS-CoV-2 RBD library to
identify mutations that prevent ADI-58124 binding. FIG. 41B
provides exemplary titration curves of indicated antibodies and
hACE2 for binding to yeast surface displayed WT SARS-CoV-2 RBD.
FIG. 41C provides exemplary flow cytometric selections of clones
from mutagenized SARS-CoV-2 RBD library with diminished ADI-58124
binding relative to WT SARS-CoV-2 RBD. FIG. 41D provides a graph
showing binding to RBD clones with single amino acid substitutions
by indicated antibodies and hACE2 relative to WT RBD. FIG. 41E
provides a heat map showing all of the RBD mutations identified
from yeast surface display selections that specifically abrogate
binding of ADI-58120, ADI-58124, ADI-58125, ADI-58126, ADI-58128,
CR3022, REGN10933, REGN10987, JS016, S309, and/or hACE2. Values
indicate percent antibody or hACE2 binding to the mutant SARS-CoV-2
RBD relative to the WT SARS-CoV-2 RBD, assessed at their respective
EC.sub.50 concentrations for the WT RBD construct. FIG. 41F
provides a heat map essentially same as the heat map in FIG. 41H
but only showing RBD mutations that specifically abrogate binding
of ADI-58120, ADI-58124, ADI-58125, ADI-58126, and/or ADI-58128
(top) or of ADI-58124 (bottom). FIG. 41G provides a protein
sequence alignment of representative sarbecovirus RBDs with
sequences colored by percentage sequence identity and conservation
shown as a bar plot. Positions delineating the receptor binding
motif are based on the SARS-CoV-2 RBD and residues determined to be
important for ADI-58124 binding based on fine epitope mapping data
shown in FIG. 41D are denoted by star.
[0263] FIGS. 41H-41J provide results from the escape mutant
analyses in Example 27. The ability of ADI-58120 (FIG. 41H (left)),
ADI-58124 (FIG. 41H (right)), ADI-58126 (FIG. 41I (left)),
ADI-58128 (FIG. 41I (right)), and ADI-58130 (FIG. 41J) to inhibit
infection of Vero cells by rVSV comprising indicated SARS-CoV-2 S
protein mutants was tested. Increased IC50 indicates that the
indicated mutation in the S protein conferred resistance.
[0264] FIGS. 42A-42H provide results from the competitive binding
analyses in Example 28. FIGS. 42A-42B provide exemplary bio-layer
interferometry (BLI) sensograms showing competition between
respective antibodies with ACE2 in binding to SARS-CoV-2 S protein.
ADI-26140 was used as a negative control. A rise in amplitude in
the second step (to the right of the vertical line in the
sensorgram) indicates an available binding site for ACE2 binding.
FIGS. 42C-42G provide BLI graphs of SARS-CoV-2 S trimer in solution
loading onto an anti-heavy chain probe preloaded with respective
1.sup.st antibodies, followed by loading with respective second
antibodies in solution, demonstrating competitive or
non-competitive binding between the 1.sup.st and 2.sup.nd
antibodies. A rise in amplitude in the second step indicates no
competition between the 1.sup.st and 2.sup.nd antibodies, and no
rise in amplitude in the second step indicates competition between
the 1.sup.st and 2.sup.nd antibodies. FIG. 42H provides a summary
of competition between indicated antibodies, or between an
indicated antibody and hACE2, based on the competitive binding
analyses in Example 28.
[0265] FIGS. 43A-43C provide binding of ADI-58124 and ADI-58125 to
different Fc gamma receptors (FIG. 43A), human or cynomolgus FcRn
(FIG. 43B), and human C1q (FIG. 43C). FIG. 43C depicts that Fc
engineering of ADI-58124 resulted in ADI-58125 with improved
binding to human and cynomolgus FcRn at low pH. In particular, the
expected half-life in humans for ADI-58124 was about 20-25 days,
while the expected half-life for ADI-58125 in humans is about
70-115 days.
[0266] FIGS. 43D-43E demonstrates that ADI-58124 and ADI-58125
trigger Fc-mediated effector functions. The indicated antibodies
were assessed for the ability to induce Fc-mediated effector
functions against RBD-coated targets at varying concentrations. In
FIG. 43E, primary human NK cells were analyzed for surface
expression of CD107a, indicating degranulation (top left), and the
production of IFN.gamma. (top center) or TNF.alpha. (top right)
following incubation with antibody-RBD immune complexes for 5
hours. Antibody-mediated phagocytosis of RBD-coated fluorescent
beads by differentiated HL-60 neutrophils (bottom left) or THP-1
monocytes (bottom center) was measured following incubation with
immune complexes for 18 hours. Antibody-mediated complement
deposition was measured by detection of complement component C3
onto RBD-coated fluorescent beads following incubation of guinea
pig complement with immune complexes for 20 minutes (bottom right).
In FIG. 43E, the area under the curve (AUC) in the three graphs
(ADCD, ADCP, and ADNKA CD107a) shown in FIG. 43D was calculated as
the sum of the products of response x concentration using GraphPad
Prism.
[0267] FIG. 43F provides comparison of ADI-58124 and ADI-58125 in
the ability to induce ADCC. In the top panel, SARS-CoV-2 S-coated
plates were used to evaluate ADI-58124 and ADI-58125 and negative
control antibody to assess CD16A activation of Jurkat-Luria cells.
The lower dotted line represents the baseline signal of cells and
media online (no antibody). The upper dotted line represents
positive control signal from a cell stimulation cocktail plus
ionomycin. In the bottom panel, SARS-CoV2 RBD-coated plates used in
the ADCC assay.
[0268] FIGS. 44A-44D demonstrate that ADI-58125 protects mice from
SARS-CoV- and SARS-CoV-2-associated viral diseases, as evaluated in
Example 30. FIG. 44A and FIG. 44B show efficacy of prophylactic
treatment with ADI-58125 in SARS-CoV-MA15 and SARS-CoV-2-MA10
challenge models, respectively. A single dose of ADI-58125 or sham
treatment were delivered intraperitoneally 12 hours prior to
infection. Mouse body weight and respiratory function were
monitored for 4 days. Gross lung hemorrhage scores were determined
on day 2 (MA15) or day 4 (MA10) post-infection and lung viral
titers were measured on day 2 and day 4 post-infection. FIG. 44C
provides effects by therapeutic treatment with ADI-58125 or sham
treatment at 12 hours post-SARS-CoV-2-MA15-infection. Mouse body
weight, respiratory function, gross hemorrhage scores (day 2), and
lung viral titers (days 2 and 4) were assessed as described above.
Statistical comparisons were made using Mann-Whitney U tests or
two-sided t-tests with Holm-Sidak corrections for multiple
comparisons (*P<0.05, **P<0.01; ***P<0.001). Dotted lines
indicate the limit of detection. FIG. 44D shows exemplary
neutralizing activity by ADI-58124 against SARS-CoV-MA
("SARS1MA")-nLuc or WT or mouse adapted SARS-CoV-2 ("SARS2"), the
virus used in the in vivo study. In the top panel, mean and SEM of
the luciferase signal are expressed as percentage inhibition or
neutralization for ADI-58125 and SARS-CoV MA-nLuc. In the bottom
panel, mean and SEM of the luciferase signal are expressed as
percentage inhibition or neutralization for ADI-58125 and
SARS-CoV-2 MA-nLuc. A table showing IC50s for ADI-58125 in the
neutralization curves for SARS1-MA-nLuc and SARS2-MA2-nLuc is also
provided.
[0269] FIGS. 45A-45B provide results from the studies on potential
antibody-dependent enhancement (ADE) in Example 31.
Luciferase-labeled reporter viral particles were culture with
phagocytes, THP-1 cells (FIG. 45A) or Raji cells (FIG. 45B), in the
presence of varied concentrations of respective test antibodies.
Increase in the luciferase signal indicates uptake of viral
particles by phagocytes.
[0270] FIG. 45C provide results from the studies on potential
antibody-dependent enhancement (ADE) for ADI-58124 and
ADI-58125.
[0271] FIG. 46 depicts the serum concentration of ADI-58125 and the
predicted serum neutralizing titers following a single 300 mg
intramuscular (IM) dose.
[0272] FIG. 47A depicts the serum concentration of ADI-58125 and
the upper and lower respiratory ELF concentrations following a
single dose of 600 mg IM.
[0273] FIG. 47B depicts the serum concentration of ADI-58125 and
the upper and lower respiratory ELF concentrations following a
single 1200 mg intravenous (IV) dose over 1 hour.
[0274] FIG. 47C depicts the serum concentration of LY-CoV555 and
the upper and lower respiratory ELF concentrations following a
single dose of 700 mg IV over 1 hour.
[0275] FIG. 47D depicts the serum concentration of REGN10987 and
the upper and lower respiratory ELF concentrations following a
single dose of 1200 mg IV over 1 hour.
[0276] FIG. 48A depicts the viral load following a single dose of
600 mg IM ADI-58125 and 2400 mg IV REGN-COV2.
[0277] FIG. 48B depicts the viral load following a single dose of
1200 mg IV ADI-58125 and 2400 mg IV REGN-COV2.
[0278] FIG. 48C depicts the viral load change relative to REGN-COV2
following a single dose of 600 mg IM ADI-58125 and 2400 mg IV
REGN-COV2.
[0279] FIG. 48D depicts the viral load change relative to REGN-COV2
following a single dose of 1200 mg IV ADI-58125 and 2400 mg IV
REGN-COV2.
[0280] FIG. 49A depicts the mean serum concentration of ADI-58125
after IV fusion and IM administration to male and female cynomolgus
monkeys. FIG. 49B depicts the pharmacokinetic parameters of
ADI-58125 in male and female Cynomolgus monkeys.
[0281] FIGS. 50A-50C depict that ADI-58122 and ADI-58125 potently
neutralize UK (B.1.1.7), South African (B.1.351) and/or Brazilian
(P.1) SARS-CoV-2 variants. FIG. 50A shows the IC50 values for
selected antibodies against the indicated SARS-CoV-2 variants. FIG.
50B depicts the maximal neutralization level for ADI-58122,
ADI-58125, REGN10987 and S309 against the indicated SARS-CoV-2
variants. FIG. 50C depicts the maximal neutralization level for
selected antibodies against the indicated SARS-CoV-2 variants.
[0282] FIG. 51 depicts that neutralization IC50 for selected
antibodies against the Victoria, UK (B.1.1.7) and South African
(B.1.351) SARS-CoV-2 variants.
[0283] FIG. 52A depicts that ADI-58125 retains high binding
affinity to the RBDs of the B.1.1.7 (UK), B.1.351 (South African)
and B.1.1.128 (Brazilian) variants. FIG. 52B depicts that ADI-58125
retains high binding affinity to the RBDs of the B.1.1.7 (UK),
B.1.351 (South African), B.1.1.128 (Brazilian) and B.1.429
(southern California) variants.
[0284] FIG. 53A is a schematic representation for the in vivo
efficacy study in hamsters. Hamsters were treated with a range of
ADI-58125 doses (9.25-2000 .mu.g) or control mAb 24 hours prior to
challenge with SARS-2/WA-1 to evaluate prophylactic efficacy of
ADI-58125.
[0285] FIGS. 53B and 53C depict the prophylactic efficacy of
ADI-58125 in the Syrian hamster model of SARS-CoV-2 infection as
assessed by viral load (plaque assay, genomic RT-PCR, subgenomic
RT-PCR), body weight, and histopathology. FIG. 53B depicts the
SARS-CoV-2 lung viral loads on Days 3 and 6 (by plaque assay), and
FIG. 53C depicts the changes from baseline in weight on Day 6.
[0286] FIG. 54A is a schematic representation for the in vivo
efficacy study in non-human primates (NHP). Rhesus macaques were
treated with ADI-58125 at 5 mg/kg or 25 mg/kg, or control mAb (25
mg/kg) 3 days prior to challenge with SARS-2/WA-1 to evaluate
prophylactic efficacy of ADI-58125.
[0287] FIG. 54B depicts the prophylactic efficacy of ADI-58125 in
the NHP model of SARS-CoV-2 infection: impact on genomic (left
panels) and sub-genomic (right panels) viral RNA in NP and BAL
samples. BAL, bronchoalveolar lavage; NP, nasopharyngeal; RT-PCR,
reverse transcription-polymerase chain reaction.
DETAILED DESCRIPTION
A. Definitions
[0288] It is to be understood that this disclosure is not limited
to the particular methodology, protocols, cell lines, animal
species or genera, and reagents described, as such may vary. It is
also to be understood that the terminology used herein is for the
purpose of describing particular embodiments only and is not
intended to limit the scope of the present disclosure, which will
be limited only by the appended claims. As used herein the singular
forms "a", "and", and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a cell" includes a plurality of such cells and reference to "the
protein" includes reference to one or more proteins and equivalents
thereof known to those skilled in the art, and so forth. All
technical and scientific terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art to which
this disclosure belongs unless clearly indicated otherwise.
[0289] Spike protein (S protein): As used herein, unless stated
otherwise S protein includes any coronavirus form of S protein. The
term coronavirus S protein ("CoV-S") is used to describe the S
protein of any coronaviruses. In particular, the "SARS-CoV-S" and
"SARS-CoV-2-S" encompass the S protein of SARS-CoV and of
SARS-CoV-2. SEQ ID NO: 1 is an exemplary polypeptide sequence of
SARS-CoV-S, comprising 1288 amino acids (Accession #PDB: 6VSB_B).
SEQ ID NO: 5 is an exemplary polypeptide sequence of SARS-CoV-2-S,
comprising 1273 amino acids (GenBank: QHD43416.1). SEQ ID NO: 2
(3864 nucleotides) encodes the SARS-CoV-S(SEQ ID NO: 1) and SEQ ID
NO: 6 (3822 nucleotides, NC_045512:21563.25384, also see the
corresponding region of GenBank: MN908947) encodes SARS-CoV-2-S(SEQ
ID NO: 5).
[0290] In some embodiments, the "SARS-CoV-S" and "SARS-CoV-2-S"
encompass any mutants, splice variants, isoforms, orthologs,
homologs, and variants of SEQ ID Nos 1 and 5. In some embodiments,
the CoV-S comprises a polypeptide sequence having at least 85%,
90%, 95%, 96%, 97%, 98%, or 99% identity to either SEQ ID NO:1 or
SEQ ID NO:5.
[0291] "Effective treatment or prevention of CoV infection" herein
refers to eliminating CoV from the subject or preventing the
expansion of CoV in the subject or eliminating or reducing the
symptoms such as fever, cough, shortness of breath, runny nose,
congestion, conjunctivitis, and/or gastrointestinal symptoms after
administration of an effective amount of an anti-CoV-S antibody or
antigen-binding fragment thereof. In some instances, effective
treatment may eliminate the need for the subject to be placed on a
ventilator or reduce the time the subject needs to be on a
ventilator. The treatment may be effected as a monotherapy or in
association with another active agent such as an antiviral agent or
anti-inflammatory agent by way of example.
[0292] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. For purposes of this
disclosure, beneficial or desired clinical results include, but are
not limited to, one or more of the following: improvement in any
aspect of COV-S-related conditions such as fever or cough. For
example, in the context of CoV infection treatment this includes
lessening severity, alleviation of fever, cough, shortness of
breath, and other associated symptoms, reducing frequency of
recurrence, increasing the quality of life of those suffering from
the CoV-related symptoms, and decreasing dose of other medications
required to treat the CoV-related symptoms. Other associated
symptoms include, but are not limited to, diarrhea, conjunctivitis,
loss of smell, and loss of taste. Still other symptoms which may be
alleviated or prevented include inflammation, cytokine storm and/or
sepsis.
[0293] "Reducing incidence" or "prophylaxis" or "prevention" means
any of reducing severity for a particular disease, condition,
symptom, or disorder (the terms disease, condition, and disorder
are used interchangeably throughout the application). Reduction in
severity includes reducing drugs and/or therapies generally used
for the condition by, for example, reducing the need for, amount
of, and/or exposure to drugs or therapies. Reduction in severity
also includes reducing the duration, and/or frequency of the
particular condition, symptom, or disorder (including, for example,
delaying or increasing time to next episodic attack in an
individual). This further includes eliminating the need for the
subject to be placed on a ventilator or reducing the time the
subject needs to be on a ventilator.
[0294] "Ameliorating" one or more symptoms of CoV infection-related
conditions means a lessening or improvement of one or more symptoms
of the condition, e.g., fever or cough or shortness of breath as
compared to not administering an anti-CoV-S antagonist antibody.
"Ameliorating" also includes shortening or reduction in duration of
a symptom. Again, this may include eliminating the need for the
subject to be placed on a ventilator or reducing the time the
subject needs to be on a ventilator.
[0295] As used herein, "controlling CoV-related symptom" or
"controlling" another CoV-S-related condition refers to maintaining
or reducing severity or duration of one or more symptoms of the
condition (as compared to the level before treatment). For example,
the duration or severity or frequency of symptoms is reduced by at
least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or
100% in the individual as compared to the level before treatment.
The reduction in the duration or severity, or frequency of symptoms
can last for any length of time, e.g., 2 weeks, 4 weeks (1 month),
8 weeks (2 months), 16 weeks (3 months), 4 months, 5 months, 6
months, 9 months, 12 months, etc.
[0296] As used therein, "delaying" the development of a
CoV-S-related condition such as shortness of breath, bronchitis, or
pneumonia e.g., interstitial), means to defer, hinder, slow,
retard, stabilize, and/or postpone progression of the condition or
disease. This delay can be of varying lengths of time, depending on
the history of the condition or disease and/or individuals being
treated. As is evident to one skilled in the art, a sufficient or
significant delay can, in effect, encompass prevention, in that the
individual does not develop symptoms. A method that "delays"
development of the symptom is a method that reduces probability of
developing the symptom in a given time frame and/or reduces extent
of the symptoms in a given time frame, when compared to not using
the method. Such comparisons are typically based on clinical
studies, using a statistically significant number of subjects.
[0297] "Development" or "progression" of a CoV-related condition
such as cough or fever means initial manifestations and/or ensuing
progression of the disorder. Development of cough or fever can be
detectable and assessed using standard clinical techniques as well
known in the art. However, development also refers to progression
that may be undetectable. For purpose of this disclosure,
development, or progression refers to the biological course of the
symptoms. "Development" includes occurrence, recurrence, and onset.
As used herein "onset" or "occurrence" of a condition includes
initial onset and/or recurrence.
[0298] As used herein, an "effective dosage" or "effective amount"
of drug, compound, or pharmaceutical composition is an amount
sufficient to effect beneficial or desired results. For
prophylactic use, beneficial or desired results include results
such as eliminating or reducing the risk, lessening the severity,
or delaying the outset of the disease, including biochemical,
histological, and/or behavioral symptoms of the disease, its
complications and intermediate pathological phenotypes presenting
during development of the disease. For therapeutic use, beneficial
or desired results include clinical results such as reducing
symptom intensity, duration, or frequency, and decreasing one or
more symptoms resulting from CoV infection, including its
complications and intermediate pathological phenotypes presenting
during development of the disease, increasing the quality of life
of those suffering from the disease, decreasing the dose of other
medications required to treat the disease, enhancing effect of
another medication, and/or delaying the progression of the disease
of patients, eliminating the need for the subject to be placed on a
ventilator or reducing the time the subject needs to be on a
ventilator.
[0299] An effective dosage can be administered in one or more
administrations. For purposes of this disclosure, an effective
dosage of drug, compound, or pharmaceutical composition is an
amount sufficient to accomplish prophylactic or therapeutic
treatment either directly or indirectly. As is understood in the
clinical context, an effective dosage of a drug, compound, or
pharmaceutical composition may or may not be achieved in
conjunction with another drug, compound, or pharmaceutical
composition. Thus, an "effective dosage" may be considered in the
context of administering one or more therapeutic agents, and a
single agent may be considered to be given in an effective amount
if, in conjunction with one or more other agents, a desirable
result may be or is achieved.
[0300] A "suitable host cell" or "host cell" generally includes any
cell wherein the subject anti-CoV-S antibodies and antigen-binding
fragments thereof can be produced recombinantly using techniques
and materials readily available. For example, the anti-CoV-S
antibodies and antigen-binding fragments thereof of the present
disclosure can be produced in genetically engineered host cells
according to conventional techniques. Suitable host cells are those
cell types that can be transformed or transfected with exogenous
DNA and grown in culture, and include bacteria, fungal cells (e.g.,
yeast), and cultured higher eukaryotic cells (including cultured
cells of multicellular organisms), particularly cultured mammalian
cells, e.g., human or non-human mammalian cells. In an exemplary
embodiment these antibodies may be expressed in CHO cells.
Techniques for manipulating cloned DNA molecules and introducing
exogenous DNA into a variety of host cells are disclosed by
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.,
Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press
(1989), and Current Protocols in Molecular Biology, Ausubel et al.,
editors, New York, N.Y.: Green and Wiley and Sons (1993).
[0301] In some exemplary embodiments the antibodies may be
expressed in mating competent yeast, e.g., any haploid, diploid or
tetraploid yeast that can be grown in culture. Yeast useful in
fermentation expression methods may exist in a haploid, diploid, or
other polyploid form.
[0302] A "selectable marker" herein refers to a gene or gene
fragment that confers a growth phenotype (physical growth
characteristic) on a cell receiving that gene as, for example
through a transformation event. The selectable marker allows that
cell to survive and grow in a selective growth medium under
conditions in which cells that do not receive that selectable
marker gene cannot grow. Selectable marker genes generally fall
into several types, including positive selectable marker genes such
as a gene that confers on a cell resistance to an antibiotic or
other drug, temperature when two temperature sensitive ("ts")
mutants are crossed or a ts mutant is transformed; negative
selectable marker genes such as a biosynthetic gene that confers on
a cell the ability to grow in a medium without a specific nutrient
needed by all cells that do not have that biosynthetic gene, or a
mutagenized biosynthetic gene that confers on a cell inability to
grow by cells that do not have the wild type gene; and the
like.
[0303] An "expression vector" herein refers to DNA vectors
containing elements that facilitate manipulation for the expression
of a foreign protein within the target host cell, e.g., a
bacterial, insect, yeast, plant, amphibian, reptile, avian, or
mammalian cell, e.g., a CHO or HEK cell. Conveniently, manipulation
of sequences and production of DNA for transformation may first
performed in a bacterial host, e.g. E. coli, and usually vectors
will include sequences to facilitate such manipulations, including
a bacterial origin of replication and appropriate bacterial
selection marker. Selection markers encode proteins necessary for
the survival or growth of transformed host cells grown in a
selective culture medium. Host cells not transformed with the
vector containing the selection gene will not survive in the
culture medium. Typical selection genes encode proteins that (a)
confer resistance to antibiotics or other toxins, (b) complement
auxotrophic deficiencies, or (c) supply critical nutrients not
available from complex media. Exemplary vectors and methods for
transformation of yeast are described, for example, in Burke, D.,
Dawson, D., & Stearns, T., Methods in yeast genetics: a Cold
Spring Harbor Laboratory course manual, Plainview, N.Y.: Cold
Spring Harbor Laboratory Press (2000). Expression vectors for use
in the methods of the disclosure may include yeast or mammalian
specific sequences, including a selectable auxotrophic or drug
marker for identifying transformed host strains. A drug marker may
further be used to amplify copy number of the vector in a yeast
host cell.
[0304] The polypeptide coding sequence of interest is operably
linked to transcriptional and translational regulatory sequences
that provide for expression of the polypeptide in the desired host
cells, e.g., yeast or mammalian cells. These vector components may
include, but are not limited to, one or more of the following: an
enhancer element, a promoter, and a transcription termination
sequence. Sequences for the secretion of the polypeptide may also
be included, e.g. a signal sequence, and the like. An origin of
replication, e.g., a yeast or mammalian origin of replication, is
optional, as expression vectors may be integrated into the host
cell genome.
[0305] Nucleic acids are "operably linked" when placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a signal sequence is operably linked to DNA for a
polypeptide if it is expressed as a preprotein that participates in
the secretion of the polypeptide; a promoter or enhancer is
operably linked to a coding sequence if it affects the
transcription of the sequence. Generally, "operably linked" means
that the DNA sequences being linked are contiguous, and, in the
case of a secretory leader, contiguous and in reading frame.
However, enhancers do not have to be contiguous. Linking is
accomplished by ligation at convenient restriction sites or via a
PCR/recombination method familiar to those skilled in the art
(GATEWAY.RTM. Technology (universal method for cloning DNA);
Invitrogen, Carlsbad Calif.). If such sites do not exist, the
synthetic oligonucleotide adapters or linkers are used in
accordance with conventional practice.
[0306] Promoters are untranslated sequences located upstream (5')
to the start codon of a structural gene (generally within about 100
to 1000 bp) that control the transcription and translation of
particular nucleic acid sequences to which they are operably
linked. Such promoters fall into several classes: inducible,
constitutive, and repressible promoters (that increase levels of
transcription in response to absence of a repressor). Inducible
promoters may initiate increased levels of transcription from DNA
under their control in response to some change in culture
conditions, e.g., the presence or absence of a nutrient or a change
in temperature.
[0307] The promoter fragment may also serve as the site for
homologous recombination and integration of the expression vector
into the same site in the host cell, e.g., yeast or mammalian cell,
genome; alternatively, a selectable marker may be used as the site
for homologous recombination. Suitable promoters for use in
different eukaryotic and prokaryotic cells are well known and
commercially available.
[0308] The polypeptides of interest may be produced recombinantly
not only directly, but also as a fusion polypeptide with a
heterologous polypeptide, e.g. a signal sequence or other
polypeptide having a specific cleavage site at the N-terminus of
the mature protein or polypeptide. In general, the signal sequence
may be a component of the vector, or it may be a part of the
polypeptide coding sequence that is inserted into the vector. The
heterologous signal sequence selected preferably is one that is
recognized and processed through one of the standard pathways
available within the host cell, e.g., a mammalian cell, an insect
cell, or a yeast cell. Additionally, these signal peptide sequences
may be engineered to provide for enhanced secretion in expression
systems. Secretion signals of interest also include mammalian and
yeast signal sequences, which may be heterologous to the protein
being secreted, or may be a native sequence for the protein being
secreted. Signal sequences include pre-peptide sequences, and in
some instances may include propeptide sequences. Many such signal
sequences are known in the art, including the signal sequences
found on immunoglobulin chains, e.g., K28 preprotoxin sequence,
PHA-E, FACE, human MCP-1, human serum albumin signal sequences,
human Ig heavy chain, human Ig light chain, and the like. For
example, see Hashimoto et. al., Protein Eng., 11(2):75 (1998); and
Kobayashi et. al., Therapeutic Apheresis, 2(4):257 (1998)).
[0309] Transcription may be increased by inserting a
transcriptional activator sequence into the vector. These
activators are cis-acting elements of DNA, usually about from 10 to
300 bp, which act on a promoter to increase its transcription.
Transcriptional enhancers are relatively orientation and position
independent, having been found 5' and 3' to the transcription unit,
within an intron, as well as within the coding sequence itself. The
enhancer may be spliced into the expression vector at a position 5'
or 3' to the coding sequence, but is preferably located at a site
5' from the promoter.
[0310] Expression vectors used in eukaryotic host cells may also
contain sequences necessary for the termination of transcription
and for stabilizing the mRNA. Such sequences are commonly available
from 3' to the translation termination codon, in untranslated
regions of eukaryotic or viral DNAs or cDNAs. These regions contain
nucleotide segments transcribed as polyadenylated fragments in the
untranslated portion of the mRNA.
[0311] Construction of suitable vectors containing one or more of
the above-listed components employs standard ligation techniques or
PCR/recombination methods. Isolated plasmids or DNA fragments are
cleaved, tailored, and re-ligated in the form desired to generate
the plasmids required or via recombination methods. For analysis to
confirm correct sequences in plasmids constructed, the ligation
mixtures are used to transform host cells, and successful
transformants selected by antibiotic resistance (e.g. ampicillin or
Zeocin) where appropriate. Plasmids from the transformants are
prepared, analyzed by restriction endonuclease digestion, and/or
sequenced.
[0312] As an alternative to restriction and ligation of fragments,
recombination methods based on specific attachment ("att") sites
and recombination enzymes may be used to insert DNA sequences into
a vector. Such methods are described, for example, by Landy, Ann.
Rev. Biochem., 58:913-949 (1989); and are known to those of skill
in the art. Such methods utilize intermolecular DNA recombination
that is mediated by a mixture of lambda and E. coli-encoded
recombination proteins. Recombination occurs between att sites on
the interacting DNA molecules. For a description of att sites see
Weisberg and Landy, Site-Specific Recombination in Phage Lambda, in
Lambda II, p. 211-250, Cold Spring Harbor, N.Y.: Cold Spring Harbor
Press (1983). The DNA segments flanking the recombination sites are
switched, such that after recombination, the att sites are hybrid
sequences comprised of sequences donated by each parental vector.
The recombination can occur between DNAs of any topology.
[0313] Att sites may be introduced into a sequence of interest by
ligating the sequence of interest into an appropriate vector;
generating a PCR product containing att B sites through the use of
specific primers; generating a cDNA library cloned into an
appropriate vector containing att sites; and the like.
[0314] Folding, as used herein, refers to the three-dimensional
structure of polypeptides and proteins, where interactions between
amino acid residues act to stabilize the structure. While
non-covalent interactions are important in determining structure,
usually the proteins of interest will have intra- and/or
intermolecular covalent disulfide bonds formed by two cysteine
residues. For naturally occurring proteins and polypeptides or
derivatives and variants thereof, the proper folding is typically
the arrangement that results in optimal biological activity, and
can conveniently be monitored by assays for activity, e.g. ligand
binding, enzymatic activity, etc.
[0315] In some instances, for example where the desired product is
of synthetic origin, assays based on biological activity will be
less meaningful. The proper folding of such molecules may be
determined on the basis of physical properties, energetic
considerations, modeling studies, etc.
[0316] The expression host may be further modified by the
introduction of sequences encoding one or more enzymes that enhance
folding and disulfide bond formation, i.e. foldases, chaperonins,
etc. Such sequences may be constitutively or inducibly expressed in
the host cell, using vectors, markers, etc. as known in the art.
Preferably the sequences, including transcriptional regulatory
elements sufficient for the desired pattern of expression, are
stably integrated in the yeast genome through a targeted
methodology.
[0317] For example, the eukaryotic protein disulfide isomerase
("PDI") is not only an efficient catalyst of protein cysteine
oxidation and disulfide bond isomerization, but also exhibits
chaperone activity. Co-expression of PDI can facilitate the
production of active proteins having multiple disulfide bonds. Also
of interest is the expression of immunoglobulin heavy chain binding
protein ("BIP"); cyclophilin; and the like.
[0318] Cultured mammalian cells are exemplary hosts for production
of the disclosed anti-CoV-S antibodies and antigen-binding
fragments thereof. As mentioned CHO cells are particularly suitable
for expression of antibodies. Many procedures are known in the art
for manufacturing monoclonal antibodies in mammalian cells. (See,
Galfre, G. and Milstein, C., Methods Enzym., 73:3-46, 1981; Basalp
et al., Turk. J. Biol., 24:189-196, 2000; Wurm, F. M., Nat.
Biotechnol., 22:1393-1398, 2004; and Li et al., mAbs, 2(5):466-477,
2010). As mentioned in further detail infra, common host cell lines
employed in mammalian monoclonal antibody manufacturing schemes
include, but are not limited to, human embryonic retinoblast cell
line PER.C6.RTM. (Crucell N.V., Leiden, The Netherlands), NSO
murine myeloma cells (Medical Research Council, London, UK), CV1
monkey kidney cell line, 293 human embryonic kidney cell line, BHK
baby hamster kidney cell line, VERO African green monkey kidney
cell line, human cervical carcinoma cell line HELA, MDCK canine
kidney cells, BRL buffalo rat liver cells, W138 human lung cells,
HepG2 human liver cells, MMT mouse mammary tumor cells, TRI cells,
MRC5 cells, Fs4 cells, myeloma or lymphoma cells, or Chinese
Hamster (Cricetulus griseus) Ovary (CHO) cells, and the like. Many
different subclones or sub-cell lines of CHO cells known in the art
that are useful and optimized for production of recombinant
monoclonal antibodies, such as the DP12 (CHO K1 dhfr-) cell line,
NSO cells are a non-Ig secreting, non-light chain-synthesizing
subclone of NS-1 cells that are resistant to azaguanine. Other
Chinese Hamster and CHO cells are commercially available (from
ATCC, etc.), including CHO-DXB11 (CHO-DUKX), CHO-pro3, CHO-DG44,
CHO 1-15, CHO DP-12, Lec2, M1WT3, Lec8, pgsA-745, and the like, all
of which are genetically altered to optimize the cell line for
various parameters. Monoclonal antibodies are commonly manufactured
using a batch fed method whereby the monoclonal antibody chains are
expressed in a mammalian cell line and secreted into the tissue
culture medium in a bioreactor. Medium (or feed) is continuously
supplied to the bioreactor to maximize recombinant protein
expression. Recombinant monoclonal antibody is then purified from
the collected media. In some circumstances, additional steps are
needed to reassemble the antibodies through reduction of disulfide
bonds, etc. Such production methods can be scaled to be as large as
10,000 L in a single batch or more. It is now routine to obtain as
much as 20 pg/cell/day through the use of such cell lines and
methodologies, providing titers as high as 10 g/L or more,
amounting to 15 to 100 kg from bioreactors of 10 kL to 25 kL. (Li
et al., 2010). Various details of this production methodology,
including cloning of the polynucleotides encoding the antibodies
into expression vectors, transfecting cells with these expression
vectors, selecting for transfected cells, and expressing and
purifying the recombinant monoclonal antibodies from these cells
are provided below.
[0319] For recombinant production of an anti-CoV-S antibody or
antigen-binding fragment in mammalian cells, nucleic acids encoding
the antibody or fragment thereof are generally inserted into a
replicable vector for further cloning (amplification of the DNA) or
for expression. DNA encoding the antibody is readily isolated or
synthesized using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
DNAs encoding the heavy and light chains of the antibody). The
vector components generally include, but are not limited to, one or
more of the following: a signal sequence, an origin of replication,
one or more marker genes, an enhancer element, a promoter, and a
transcription termination sequence. Selection of promoters,
terminators, selectable markers, vectors, and other elements is a
matter of routine design within the level of ordinary skill in the
art. Many such elements are known in the art and are available
through commercial suppliers.
[0320] The antibodies of this disclosure may be produced
recombinantly not only directly, but also as a fusion polypeptide
with a heterologous polypeptide, which is preferably a signal
sequence or other polypeptide having a specific cleavage site at
the N-terminus of the mature protein or polypeptide. The homologous
or heterologous signal sequence selected preferably is one that is
recognized and processed (i.e., cleaved by a signal peptidase) by
the host cell. In mammalian cell expression, mammalian signal
sequences as well as viral secretory leaders, for example, the
herpes simplex gD signal, are available.
[0321] Such expression vectors and cloning vectors will generally
contain a nucleic acid sequence that enables the vector to
replicate in one or more selected host cells. Typically, in cloning
vectors this sequence is one that enables the vector to replicate
independently of the host chromosomal DNA, and includes origins of
replication or autonomously replicating sequences. Such sequences
are well known for a variety of bacteria, yeast, and viruses, e.g.,
the origin of replication from the plasmid pBR322 is suitable for
most Gram-negative bacteria, the 2 mu plasmid origin is suitable
for yeast, and various viral origins (Simian Virus 40 ("SV40"),
polyoma, adenovirus, vesicular stomatitis virus ("VSV"), or bovine
papillomavirus ("BPV") are useful for cloning vectors in mammalian
cells. Generally, the origin of replication component is not needed
for mammalian expression vectors (the SV40 origin may typically be
used only because it contains the early promoter).
[0322] These vectors will also typically contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli.
[0323] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Drug selection is generally used to select
for cultured mammalian cells into which foreign DNA has been
inserted. Such cells are commonly referred to as "transfectants".
Cells that have been cultured in the presence of the selective
agent and are able to pass the gene of interest to their progeny
are referred to as "stable transfectants." Examples of such
dominant selection use the drugs neomycin, mycophenolic acid, and
hygromycin. An exemplary selectable marker is a gene encoding
resistance to the antibiotic neomycin. Selection is carried out in
the presence of a neomycin-type drug, such as G-418 or the like.
Those cells that are successfully transformed with a heterologous
gene produce a protein conferring drug resistance and thus survive
the selection regimen.
[0324] Selection systems can also be used to increase the
expression level of the gene of interest, a process referred to as
"amplification." Amplification of transfectants typically occurs by
culturing the cells in the presence of a low level of the selective
agent and then increasing the amount of selective agent to select
for cells that produce high levels of the products of the
introduced genes. Exemplary suitable selectable markers for
mammalian cells are those that enable the identification of cells
competent to take up the antibody nucleic acid, such as
dihydrofolate reductase ("DHFR"), thymidine kinase,
metallothionein-I and -II, preferably primate metallothionein
genes, adenosine deaminase, ornithine decarboxylase, etc.
[0325] For example, an amplifiable selectable marker for mammalian
cells is dihydrofolate reductase, which confers resistance to
methotrexate. Other drug resistance genes (e.g. hygromycin
resistance, multi-drug resistance, puromycin acetyltransferase) can
also be used. Cells transformed with the DHFR selection gene are
first identified by culturing all of the transformants in a culture
medium that contains methotrexate ("MTX"), a competitive antagonist
of DHFR. An appropriate host cell when wild-type DHFR is employed
is the Chinese hamster ovary ("CHO") cell line deficient in DHFR
activity.
[0326] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding antibody, wild-type DHFR protein, and another
selectable marker such as aminoglycoside 3'-phosphotransferase
("APH") can be selected by cell growth in medium containing a
selection agent for the selectable marker such as an
aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G-418.
See U.S. Pat. No. 4,965,199.
[0327] These vectors may comprise an enhancer sequence that
facilitates transcription of a DNA encoding the antibody. Many
enhancer sequences are known from mammalian genes (for example,
globin, elastase, albumin, alpha-fetoprotein, and insulin). A
frequently used enhancer is one derived from a eukaryotic cell
virus. Examples thereof include the SV40 enhancer on the late side
of the replication origin (bp 100-270), the cytomegalovirus early
promoter enhancer, the polyoma enhancer on the late side of the
replication origin, and adenovirus enhancers (See also Yaniv,
Nature, 297:17-18 (1982) on enhancing elements for activation of
eukaryotic promoters). The enhancer may be spliced into the vector
at a position 5' or 3' to the antibody-encoding sequence, but is
preferably located at a site 5' from the promoter.
[0328] Expression and cloning vectors will also generally comprise
a promoter that is recognized by the host organism and is operably
linked to the antibody nucleic acid. Promoter sequences are known
for eukaryotes. Virtually all eukaryotic genes have an AT-rich
region located approximately 25 to 30 bases upstream from the site
where transcription is initiated. Another sequence found 70 to 80
bases upstream from the start of transcription of many genes is a
CNCAAT region where N may be any nucleotide. At the 3' end of most
eukaryotic genes is an AATAAA sequence that may be the signal for
addition of the poly A tail to the 3' end of the coding sequence.
All of these sequences are suitably inserted into eukaryotic
expression vectors.
[0329] Antibody transcription from vectors in mammalian host cells
may be controlled, for example, by promoters obtained from the
genomes of viruses such as polyoma virus, fowlpox virus, adenovirus
(such as Adenovirus 2), BPV, avian sarcoma virus, cytomegalovirus,
a retrovirus, hepatitis-B virus, and most preferably SV40, from
heterologous mammalian promoters, e.g., the actin promoter or an
immunoglobulin promoter, from heat-shock promoters, provided such
promoters are compatible with the host cell systems.
[0330] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the BPV as a vector is disclosed in U.S. Pat.
No. 4,419,446. A modification of this system is described in U.S.
Pat. No. 4,601,978. See also Reyes et al., Nature, 297:598-601
(1982) on expression of human beta-interferon cDNA in mouse cells
under the control of a thymidine kinase promoter from herpes
simplex virus. Alternatively, the Rous sarcoma virus long terminal
repeat can be used as the promoter.
[0331] Strong transcription promoters can be used, such as
promoters from SV40, cytomegalovirus, or myeloproliferative sarcoma
virus. See, e.g., U.S. Pat. No. 4,956,288 and U.S. Patent
Publication No. 20030103986. Other suitable promoters include those
from metallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978)
and the adenovirus major late promoter. Expression vectors for use
in mammalian cells include pZP-1, pZP-9, and pZMP21, which have
been deposited with the American Type Culture Collection, 10801
University Blvd., Manassas, Va. USA under accession numbers 98669,
98668, and PTA-5266, respectively, and derivatives of these
vectors.
[0332] Expression vectors used in eukaryotic host cells (yeast,
fungus, insect, plant, animal, human, or a nucleated cell from
other multicellular organism) will also generally contain sequences
necessary for the termination of transcription and for stabilizing
the mRNA. Such sequences are commonly available from the 5' and,
occasionally 3', untranslated regions of eukaryotic or viral DNAs
or cDNAs. These regions contain nucleotide segments transcribed as
polyadenylated fragments in the untranslated portion of the mRNA
encoding the antibody. One useful transcription termination
component is the bovine growth hormone polyadenylation region. See
WO 94/11026 and the expression vector disclosed therein.
[0333] Suitable host cells for cloning or expressing the subject
antibodies include prokaryote, yeast, or higher eukaryote cells
described above. However, interest has been greatest in vertebrate
cells, and propagation of vertebrate cells in culture has become a
routine procedure. Examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-1 (ATCC No. CRL
1650); and COS-7, ATCC CRL 1651); human embryonic kidney line (293
or 293 cells subcloned for growth in suspension culture, (ATCC No.
CRL 1573; Graham et al., J. Gen. Virol., 36:59-72 (1977)); baby
hamster kidney cells (BHK, ATCC CCL 10, ATCC No. CRL 1632; BHK 570,
ATCC No. CRL 10314); CHO cells (CHO-KI, ATCC No. CCL 61; CHO-DG44,
Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980));
mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251
(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green
monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical
carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC
CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human
lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB
8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells
(Mather et al., Annals N. Y. Acad. Sci., 383:44-68 (1982)); MRC 5
cells; FS4 cells; and a human hepatoma line (Hep G2). Additional
suitable cell lines are known in the art and available from public
depositories such as the American Type Culture Collection,
Manassas, Va.
[0334] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences as discussed supra.
[0335] The mammalian host cells used to produce the antibody of
this disclosure may be cultured in a variety of media. Commercially
available media such as Ham's F10 (Sigma-Aldrich Corporation, St.
Louis, Mo.), Minimal Essential Medium (("MEM" (Sigma-Aldrich
Corporation, St. Louis, Mo.), Roswell Park Memorial Institute-1640
medium ("RPMI-1640", Sigma-Aldrich Corporation, St. Louis, Mo.),
and Dulbecco's Modified Eagle's Medium (("DMEM" Sigma-Aldrich
Corporation, St. Louis, Mo.) are suitable for culturing the host
cells. In addition, any of the media described in Ham et al., Meth.
Enz., 58:44 (1979), Barnes et al., Anal. Biochem., 102:255 (1980),
U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or
5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Reexam No. 30,985
can be used as culture media for the host cells. Any of these media
may be supplemented as necessary with hormones and/or other growth
factors (such as insulin, transferrin, or epidermal growth factor),
salts (such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as Gentamycin drug), trace elements
(defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan. Methods of development and optimization
of media and culture conditions are known in the art (See,
Gronemeyer et al., Bioengineering, 1(4):188-212, 2014).
[0336] After culture conditions are optimized and a preferred cell
line clone is selected, these cells are cultured (either adherent
cells or suspension cultures) most typically in a batch-fed process
in a bioreactor (many models are commercially available) that
involves continuously feeding the cell culture with medium and
feed, optimized for the particular cell line chosen and selected
for this purpose. (See, Butler, M., Appl. Microbiol. Biotechnol.,
68:283-291, 2005; and Kelley, B., mAb, 1(5):443-452, 2009).
Perfusion systems are also available in which media and feed are
continuously supplied to the culture while the same volume of media
is being withdrawn from the bioreactor. (Wurm, 2004). Synthetic
media, also commercially available, are available for growing cells
in a batch-fed culture, avoiding the possibility of contamination
from outside sources, such as with the use of animal components,
such as bovine serum albumin, etc. However, animal-component-free
hydrolysates are commercially available to help boost cell density,
culture viability and productivity. (Li et al., 2010). Many studies
have been performed in an effort to optimize cell culture media,
including careful attention to head space available in roller
bottles, redox potentials during growth and expression phases,
presence of reducing agents to maintain disulfide bonds during
production, etc. (See, for instance, Hutterer et al., mAbs,
5(4):608-613, 2013; and Mullan et al., BMC Proceed., 5 (Suppl
8):P110, 2011). Various methodologies have been developed to
address the possibility of harmful oxidation during recombinant
monoclonal antibody production. (See, for example, U.S. Pat. No.
8,574,869). Cultured cells may be grown by feeding nutrients
continuously or as separately administered amounts. Often various
process parameters such as cell concentration, pH, temperature,
CO.sub.2, dO.sub.2, osmolality, amount of metabolites such as
glucose, lactate, glutamine and glutamate, and the like, are
monitored by the use of probes during the cell growth either
on-line by direct connection to calibrated analyzers or off-line by
intervention of operators. The culturing step also typically
involves ensuring that the cells growing in culture maintain the
transfected recombinant genes by any means known in the art for
cell selection.
[0337] Following fermentation, i.e., upon reaching maximum cell
growth and recombinant protein expression, the culturing step is
typically followed by a harvesting step, whereby the cells are
separated from the medium and a harvested cell culture media is
thereby obtained. (See, Liu et al., mAbs, 2(5):480-499, 2010).
Typically, various purification steps, involving column
chromatography and the like, follow culturing to separate the
recombinant monoclonal antibody from cell components and cell
culture media components. The exact purification steps needed for
this phase of the production of recombinant monoclonal antibodies
depends on the site of expression of the proteins, i.e., in the
cytosol of the cells themselves, or the more commonly preferred
route of protein excreted into the cell culture medium. Various
cell components may be separated using techniques known in the art
such as differential centrifugation techniques, gravity-based cell
settling, and/or size exclusion chromatograph/filtration techniques
that can include tangential flow micro-filtration or depth
filtration. (See, Pollock et al., Biotechnol. Bioeng., 110:206-219,
2013, and Liu et al., 2010). Centrifugation of cell components may
be achieved on a large scale by use of continuous disk stack
centrifuges followed by clarification using depth and membrane
filters. (See, Kelley, 2009). Most often, after clarification, the
recombinant protein is further purified by Protein A chromatography
due to the high affinity of Protein A for the Fc domain of
antibodies, and typically occurs using a low pH/acidification
elution step (typically the acidification step is combined with a
precautionary virus inactivation step). Flocculation and/or
precipitation steps using acidic or cationic polyelectrolytes may
also be employed to separate animal cells in suspension cultures
from soluble proteins. (Liu et al., 2010). Lastly, anion- and
cation-exchange chromatography, hydrophobic interaction
chromatograph ("HIC"), hydrophobic charge induction chromatograph
(HCIC), hydroxyapatite chromatography using ceramic hydroxyapatite
(Ca.sub.5(PO.sub.4).sub.3OH).sub.2, and combinations of these
techniques are typically used to polish the solution of recombinant
monoclonal antibody. Final formulation and concentration of the
desired monoclonal antibody may be achieved by use of
ultracentrifugation techniques. Purification yields are typically
70 to 80%. (Kelley, 2009).
[0338] The terms "desiredprotein" or "desired antibody" herein are
used interchangeably and refer generally to a parent antibody
specific to a target, i.e., CoV-S or a chimeric or humanized
antibody or a binding portion thereof derived therefrom as
described herein. The term "antibody" is intended to include any
polypeptide chain-containing molecular structure with a specific
shape that fits to and recognizes an epitope, where one or more
non-covalent binding interactions stabilize the complex between the
molecular structure and the epitope. The archetypal antibody
molecule is the immunoglobulin, and all types of immunoglobulins,
IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g. human,
rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken, other
avians, etc., are considered to be "antibodies." Examples thereof
include chimeric antibodies, human antibodies and other non-human
mammalian antibodies, humanized antibodies, single chain antibodies
(such as scFvs), camelbodies, nanobodies, IgNAR (single-chain
antibodies which may be derived from sharks, for example),
small-modular immunopharmaceuticals ("SMIPs"), and antibody
fragments such as Fabs, Fab', F(ab').sub.2, and the like (See
Streltsov et al., Protein Sci., 14(11):2901-9 (2005); Greenberg et
al., Nature, 374(6518):168-73 (1995); Nuttall et al., Mol.
Immunol., 38(4):313-26 (2001); Hamers-Casterman et al., Nature,
363(6428):446-8 (1993); Gill et al., Curr. Opin. Biotechnol.,
(6):653-8 (2006)).
[0339] For example, antibodies or antigen-binding fragments thereof
may be produced by genetic engineering. In this technique, as with
other methods, antibody-producing cells are sensitized to the
desired antigen or immunogen. The messenger RNA isolated from
antibody producing cells is used as a template to make cDNA using
PCR amplification. A library of vectors, each containing one heavy
chain gene and one light chain gene retaining the initial antigen
specificity, is produced by insertion of appropriate sections of
the amplified immunoglobulin cDNA into the expression vectors. A
combinatorial library is constructed by combining the heavy chain
gene library with the light chain gene library. This results in a
library of clones that co-express a heavy and light chain
(resembling the Fab fragment or antigen-binding fragment of an
antibody molecule). The vectors that carry these genes are
co-transfected into a host cell. When antibody gene synthesis is
induced in the transfected host, the heavy and light chain proteins
self-assemble to produce active antibodies that can be detected by
screening with the antigen or immunogen.
[0340] Antibody coding sequences of interest include those encoded
by native sequences, as well as nucleic acids that, by virtue of
the degeneracy of the genetic code, are not identical in sequence
to the disclosed nucleic acids, and variants thereof. Variant
polypeptides can include amino acid ("aa") substitutions,
additions, or deletions. The amino acid substitutions can be
conservative amino acid substitutions or substitutions to eliminate
non-essential amino acids, such as to alter a glycosylation site,
or to minimize misfolding by substitution or deletion of one or
more cysteine residues that are not necessary for function.
Variants can be designed so as to retain or have enhanced
biological activity of a particular region of the protein (e.g., a
functional domain, catalytic amino acid residues, etc). Variants
also include fragments of the polypeptides disclosed herein,
particularly biologically active fragments and/or fragments
corresponding to functional domains. Techniques for in vitro
mutagenesis of cloned genes are known. Also included in the subject
disclosure are polypeptides that have been modified using ordinary
molecular biological techniques so as to improve their resistance
to proteolytic degradation or to optimize solubility properties or
to render them more suitable as a therapeutic agent.
[0341] Chimeric antibodies may be made by recombinant means by
combining the V.sub.L and VH regions, obtained from antibody
producing cells of one species with the constant light and heavy
chain regions from another. Typically, chimeric antibodies utilize
rodent or rabbit variable regions and human constant regions, in
order to produce an antibody with predominantly human domains. The
production of such chimeric antibodies is well known in the art,
and may be achieved by standard means (as described, e.g., in U.S.
Pat. No. 5,624,659, incorporated herein by reference in its
entirety). It is further contemplated that the human constant
regions of chimeric antibodies of the disclosure may be selected
from IgG1, IgG2, IgG3, and IgG4 constant regions.
[0342] Humanized antibodies are engineered to contain even more
human-like mmunoglobulin domains, and incorporate only the
complementarity determining regions of the animal-derived antibody.
This is accomplished by carefully examining the sequence of the
hyper-variable loops of the variable regions of the monoclonal
antibody and fitting them to the structure of the human antibody
chains. Although facially complex, the process is straightforward
in practice. See, e.g., U.S. Pat. No. 6,187,287, incorporated fully
herein by reference.
[0343] In addition to entire immunoglobulins (or their recombinant
counterparts), immunoglobulin fragments comprising the epitope
binding site (e.g., Fab', F(ab').sub.2, or other fragments) may be
synthesized. "Fragment" or minimal immunoglobulins may be designed
utilizing recombinant immunoglobulin techniques. For instance, "Fv"
immunoglobulins for use in the present disclosure may be produced
by synthesizing a fused variable light chain region and a variable
heavy chain region. Combinations of antibodies are also of
interest, e.g. diabodies, which comprise two distinct Fv
specificities. In another embodiment, small molecule
immunopharmaceuticals ("SMIPs"), camelbodies, nanobodies, and IgNAR
are encompassed by immunoglobulin fragments.
[0344] Immunoglobulins and fragments thereof may be modified
post-translationally, e.g. to add effector moieties such as
chemical linkers, detectable moieties, such as fluorescent dyes,
enzymes, toxins, substrates, bioluminescent materials, radioactive
materials, chemiluminescent moieties, and the like, or specific
binding moieties, such as streptavidin, avidin, or biotin, and the
like may be utilized in the methods and compositions of the present
disclosure. Examples of additional effector molecules are provided
infra.
[0345] A polynucleotide sequence "corresponds" to a polypeptide
sequence if translation of the polynucleotide sequence in
accordance with the genetic code yields the polypeptide sequence
(i.e., the polynucleotide sequence "encodes" the polypeptide
sequence), one polynucleotide sequence "corresponds" to another
polynucleotide sequence if the two sequences encode the same
polypeptide sequence.
[0346] A "heterologous" region or domain of a DNA construct is an
identifiable segment of DNA within a larger DNA molecule that is
not found in association with the larger molecule in nature. Thus,
when the heterologous region encodes a mammalian gene, the DNA
flanking the gene usually does not flank the mammalian genomic DNA
in the genome of the source organism. Another example of a
heterologous region is a construct where the coding sequence itself
is not found in nature (e.g., a cDNA where the genomic coding
sequence contains introns or synthetic sequences having codons
different than the native gene). Allelic variations or
naturally-occurring mutational events do not give rise to a
heterologous region of DNA as defined herein.
[0347] A "coding sequence" is an in-frame sequence of codons that
correspond to or encode a protein or peptide sequence. Two coding
sequences correspond to each other if the sequences or their
complementary sequences encode the same amino acid sequences. A
coding sequence in association with appropriate regulatory
sequences may be transcribed and translated into a polypeptide. A
polyadenylation signal and transcription termination sequence will
usually be located 3' to the coding sequence. A "promoter sequence"
is a DNA regulatory region capable of initiating transcription of a
downstream (3' direction) coding sequence, and typically contain
additional sites for binding of regulatory molecules, e.g.,
transcription factors, that affect the transcription of the coding
sequence. A coding sequence is "under the control" of the promoter
sequence or "operatively linked" to the promoter when RNA
polymerase binds the promoter sequence in a cell and transcribes
the coding sequence into mRNA, which is then in turn translated
into the protein encoded by the coding sequence.
[0348] The general structure of antibodies in vertebrates now is
well understood. See Edelman, G. M., Ann. N.Y. Acad. Sci., 190:5
(1971). Antibodies consist of two identical light polypeptide
chains of molecular weight approximately 23,000 daltons (the "light
chain"), and two identical heavy chains of molecular weight
53,000-70,000 (the "heavy chain"). The four chains are joined by
disulfide bonds in a "Y" configuration wherein the light chains
bracket the heavy chains starting at the mouth of the "Y"
configuration. The "branch" portion of the "Y" configuration is
designated the F.sub.ab region; the stem portion of the "Y"
configuration is designated the Fc region. The amino acid sequence
orientation runs from the N-terminal end at the top of the "Y"
configuration to the C-terminal end at the bottom of each chain.
The N-terminal end possesses the variable region having specificity
for the antigen that elicited it, and is approximately 100 amino
acids in length, there being slight variations between light and
heavy chain and from antibody to antibody.
[0349] The variable region is linked in each chain to a constant
region that extends the remaining length of the chain and that
within a particular class of antibody does not vary with the
specificity of the antibody (i.e., the antigen eliciting it). There
are five known major classes of constant regions that determine the
class of the immunoglobulin molecule (IgG, IgM, IgA, IgD, and IgE
corresponding to .gamma., .mu., .alpha., .delta., and .epsilon.
(gamma, mu, alpha, delta, or epsilon) heavy chain constant
regions). The constant region or class determines subsequent
effector function of the antibody, including activation of
complement (see Kabat, E. A., Structural Concepts in Immunology and
Immunochemistry, 2nd Ed., p. 413-436, New York, N.Y.: Holt,
Rinehart, Winston (1976)), and other cellular responses (see
Andrews et al., Clinical Immunology, pp. 1-18, W. B. Sanders,
Philadelphia, Pa. (1980); Kohl et al., Immunology, 48:187 (1983));
while the variable region determines the antigen with which it will
react. Light chains are classified as either .kappa. (kappa) or
.lamda. (lambda). Each heavy chain class can be prepared with
either kappa or lambda light chain. The light and heavy chains are
covalently bonded to each other, and the "tail" portions of the two
heavy chains are bonded to each other by covalent disulfide
linkages when the immunoglobulins are generated either by
hybridomas or by B-cells.
[0350] The expression "variable region" or "VR" refers to the
domains within each pair of light and heavy chains in an antibody
that are involved directly in binding the antibody to the antigen.
Each heavy chain has at one end a variable region (VH) followed by
a number of constant domains. Each light chain has a variable
region (VL) at one end and a constant domain at its other end; the
constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light chain variable
domain is aligned with the variable domain of the heavy chain.
[0351] The expressions "complementarity-determining region,"
"hypervariable region," or "CDR" refer to one or more of the
hyper-variable or complementarity-determining regions ("CDRs")
found in the variable regions of light or heavy chains of an
antibody (See Kabat et al., Sequences of Proteins of Immunological
Interest, 4.sup.th ed., Bethesda, Md.: U.S. Dept. of Health and
Human Services, Public Health Service, National Institutes of
Health (1987)). These expressions include the hypervariable regions
as defined by Kabat et al., (Sequences of Proteins of Immunological
Interest, NIH Publication No. 91-3242, Bethesda, Md.: U.S. Dept. of
Health and Human Services, National Institutes of Health (1983)) or
the hypervariable loops in 3-dimensional structures of antibodies
(Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)). The CDRs in
each chain are held in close proximity by framework regions ("FRs")
and, with the CDRs from the other chain, contribute to the
formation of the antigen binding site. Within the CDRs there are
select amino acids that have been described as the selectivity
determining regions ("SDRs") that represent the critical contact
residues used by the CDR in the antibody-antigen interaction (see
Kashmiri et al., Methods, 36(1):25-34 (2005)).
[0352] An "epitope" or "binding site" is an area or region on an
antigen to which an antigen-binding peptide (such as an antibody)
specifically binds. A protein epitope may comprise amino acid
residues directly involved in the binding (also called
immunodominant component of the epitope) and other amino acid
residues, which are not directly involved in the binding, such as
amino acid residues that are effectively blocked by the
specifically antigen binding peptide (in other words, the amino
acid residue is within the "footprint" of the specifically antigen
binding peptide). The term epitope herein includes both types of
amino acid binding sites in any particular region of CoV-S, e.g.,
SARS-CoV-S or SARS-CoV-2-S, that specifically binds to an
anti-CoV-S antibody. CoV-S may comprise a number of different
epitopes, which may include, without limitation, (1) linear peptide
antigenic determinants, (2) conformational antigenic determinants
that consist of one or more non-contiguous amino acids located near
each other in a mature CoV-S conformation; and (3)
post-translational antigenic determinants that consist, either in
whole or part, of molecular structures covalently attached to a
CoV-S protein such as carbohydrate groups. In particular, the term
"epitope" includes the specific residues in a protein or peptide,
e.g., CoV-S, which are involved in the binding of an antibody to
such protein or peptide as determined by known and accepted methods
such as alanine scanning techniques or the use of various S protein
portions with varying lengths.
[0353] The phrase that an antibody (e.g., first antibody) binds
"substantially" or "at least partially" the same epitope as another
antibody (e.g., second antibody) means that the epitope binding
site for the first antibody comprises at least 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, or more of the amino acid residues on the
antigen that constitutes the epitope binding site of the second
antibody. Also, that a first antibody binds substantially or
partially the same or overlapping epitope as a second antibody
means that the first and second antibodies compete in binding to
the antigen, as described above. Thus, the term "binds to
substantially the same epitope or determinant as" a monoclonal
antibody means that an antibody "competes" with the antibody.
[0354] The phrase "binds to the same or overlapping epitope or
determinant as" an antibody of interest means that an antibody
"competes" with said antibody of interest for at least one, (e.g.,
at least 2, at least 3, at least 4, at least 5) or all residues on
CoV-S to which said antibody of interest specifically binds. The
identification of one or more antibodies that bind(s) to
substantially or essentially the same epitope as the monoclonal
antibodies described herein can be readily determined using alanine
scanning. Additionally, any one of variety of immunological
screening assays in which antibody competition can be assessed. A
number of such assays are routinely practiced and well known in the
art (see, e.g., U.S. Pat. No. 5,660,827, issued Aug. 26, 1997,
which is specifically incorporated herein by reference). It will be
understood that actually determining the epitope to which an
antibody described herein binds is not in any way required to
identify an antibody that binds to the same or substantially the
same or overlapping epitope as the monoclonal antibody described
herein.
[0355] For example, where the test antibodies to be examined are
obtained from different source animals, or are even of a different
Ig isotype, a simple competition assay may be employed in which the
control antibody is mixed with the test antibody and then applied
to a sample containing CoV-S. Protocols based upon ELISAs,
radioimmunoassays, Western blotting, and the use of BIACORE.RTM.
(GE Healthcare Life Sciences, Marlborough, Mass.) analysis are
suitable for use in such simple competition studies.
[0356] In certain embodiments, the control anti-CoV-S antibody is
pre-mixed with varying amounts of the test antibody (e.g., in
ratios of about 1:1, 1:2, 1:10, or about 1:100) for a period of
time prior to applying to the CoV-S (e.g., SARS-CoV-S or
SARS-CoV-2-S) antigen sample. In other embodiments, the control and
varying amounts of test antibody can simply be added separately and
admixed during exposure to the SARS-CoV-S or SARS-CoV-2-S antigen
sample. As long as bound antibodies can be distinguished from free
antibodies (e.g., by using separation or washing techniques to
eliminate unbound antibodies) and control antibody from the test
antibody (e.g., by using species specific or isotype specific
secondary antibodies or by specifically labeling the control
antibody with a detectable label) it can be determined if the test
antibody reduces the binding of the control antibody to the
SARS-CoV-S or SARS-CoV-2-S antigens, indicating that the test
antibody recognizes substantially the same epitope as the control
anti-CoV-S antibody. The binding of the (labeled) control antibody
in the presence of a completely irrelevant antibody (that does not
bind CoV-S) can serve as the control high value. The control low
value can be obtained by incubating the labeled control antibody
with the same but unlabeled control antibody, where competition
would occur and reduce binding of the labeled antibody. In a test
assay, a significant reduction in labeled antibody reactivity in
the presence of a test antibody is indicative of a test antibody
that recognizes substantially the same epitope, i.e., one that
competes with the labeled control antibody. For example, any test
antibody that reduces the binding of the control antibody to
SARS-CoV-S or SARS-CoV-2-S by at least about 50%, such as at least
about 60%, or more preferably at least about 70% (e.g., about
65-100%), at any ratio of test antibody between about 1:1 or 1:10
and about 1:100 is considered to be an antibody that binds to
substantially the same or overlapping epitope or determinant as the
control antibody.
[0357] Preferably, such test antibody will reduce the binding of
the control antibody to SARS-CoV-S or SARS-CoV-2-S (or another
CoV-S) antigen preferably at least about 50%, at least about 60%,
at least about 80%, or at least about 90% (e.g., about 95%) of the
binding of the control antibody observed in the absence of the test
antibody.
[0358] A simple competition assay in which a test antibody is
applied at saturating concentration to a surface onto which
SARS-CoV-S or SARS-CoV-2-S (or another CoV-S) is immobilized also
may be advantageously employed. The surface in the simple
competition assay is preferably a BIACORE.RTM. (GE Healthcare Life
Sciences, Marlborough, Mass.) chip (or other media suitable for
surface plasmon resonance ("SPR") analysis). The binding of a
control antibody that binds SARS-CoV-S or SARS-CoV-2-S to the
COV-S-coated surface is measured. This binding to the SARS-CoV-S-
or SARS-CoV-2-S-containing surface of the control antibody alone is
compared with the binding of the control antibody in the presence
of a test antibody. A significant reduction in binding to the
SARS-CoV-S- or SARS-CoV-2-S-containing surface by the control
antibody in the presence of a test antibody indicates that the test
antibody recognizes substantially the same epitope as the control
antibody such that the test antibody "competes" with the control
antibody. Any test antibody that reduces the binding of control
antibody by at least about 20% or more, at least about 40%, at
least about 50%, at least about 70%, or more, can be considered to
be an antibody that binds to substantially the same epitope or
determinant as the control antibody. Preferably, such test antibody
will reduce the binding of the control antibody to SARS-CoV-S or
SARS-CoV-2-S by at least about 50% (e.g., at least about 60%, at
least about 70%, or more). It will be appreciated that the order of
control and test antibodies can be reversed; i.e. the control
antibody can be first bound to the surface and then the test
antibody is brought into contact with the surface thereafter in a
competition assay. Preferably, the "sandwich-style" binding assay
infra is used. Alternatively, the antibody having greater affinity
for SARS-CoV-S or SARS-CoV-2-S antigen is bound to the SARS-CoV-S-
or SARS-CoV-2-S-containing surface first, as it will be expected
that the decrease in binding seen for the second antibody (assuming
the antibodies are competing) will be of greater magnitude. Further
examples of such assays are provided in e.g., Saunal and
Regenmortel, J. Immunol. Methods, 183:33-41 (1995), the disclosure
of which is incorporated herein by reference.
[0359] In addition, whether an antibody binds the same or
overlapping epitope(s) on COV-S as another antibody or the epitope
bound by a test antibody may in particular be determined using a
Western-blot based assay. In this assay a library of peptides
corresponding to the antigen bound by the antibody, the CoV-S
protein, is made, that comprise overlapping portions of the
protein, typically 10-25, 10-20, or 10-15 amino acids long. These
different overlapping amino acid peptides encompassing the CoV-S
sequence are synthesized and covalently bound to a PEPSPOTS.TM.
nitrocellulose membrane (JPT Peptide Technologies, Berlin,
Germany). Blots are then prepared and probed according to the
manufacturer's recommendations.
[0360] Essentially, the immunoblot assay then detects by
fluorometric means what peptides in the library bind to the test
antibody and thereby can identify what residues on the antigen,
i.e., COV-S, interact with the test antibody. (See U.S. Pat. No.
7,935,340, incorporated by reference herein).
[0361] Various epitope mapping techniques are known in the art. By
way of example, X-ray co-crystallography of the antigen and
antibody; NMR; SPR (e.g., at 250 or 37.degree. C.); array-based
oligo-peptide scanning (or "pepscan analysis"); site-directed
mutagenesis (e.g., alanine scanning); mutagenesis mapping;
hydrogen-deuterium exchange; phage display; and limited proteolysis
are all epitope mapping techniques that are well known in the art
(See, e.g., Epitope Mapping Protocols: Second Edition, Methods in
Molecular Biology, editors Mike Schutkowski and Ulrich Reineke,
2.sup.nd Ed., New York, N.Y.: Humana Press (2009), and Epitope
Mapping Protocols, Methods in Molecular Biology, editor Glenn
Morris, 1.sup.st Ed., New York, N.Y.: Humana Press (1996), both of
which are herein incorporated by referenced in their entirety).
[0362] The identification of one or more antibodies that bind(s) to
substantially or essentially the same epitope as the monoclonal
antibodies described herein, e.g., any one of antibodies as
described herein and in FIGS. 1, 2 and 36, can be readily
determined using any one of variety of immunological screening
assays in which antibody competition can be assessed. A number of
such assays are routinely practiced and well known in the art (see,
e.g., U.S. Pat. No. 5,660,827, issued Aug. 26, 1997, which is
incorporated herein by reference). It will be understood that
determining the epitope to which an antibody described herein binds
is not in any way required to identify an antibody that binds to
the same or substantially the same epitope as the monoclonal
antibody described herein.
[0363] For example, where the test antibodies to be examined are
obtained from different source animals, or are even of a different
Ig isotype, a simple competition assay may be employed in which the
control antibody (one of antibodies as described above and in FIGS.
1, 2 and 36, for example) is mixed with the test antibody and then
applied to a sample containing either or both SARS-CoV-S or
SARS-CoV-2-S, each of which is known to be bound by antibodies as
described above and in FIGS. 1, 2 and 36. Protocols based upon
ELISAs, radioimmunoassays, Western blotting, and BIACORE.RTM. (GE
Healthcare Life Sciences, Marlborough, Mass.) analysis (as
described in the Examples section herein) are suitable for use in
such simple competition studies.
[0364] In certain embodiments, the method comprises pre-mixing the
control antibody with varying amounts of the test antibody (e.g.,
in ratios of about 1:1, 1:2, 1:10, or about 1:100) for a period of
time prior to applying to the CoV-S antigen sample. In other
embodiments, the control and varying amounts of test antibody can
be added separately and admixed during exposure to the CoV-S
antigen sample. As long as bound antibodies can be distinguished
from free antibodies (e.g., by using separation or washing
techniques to eliminate unbound antibodies) and control antibody
from the test antibody (e.g., by using species specific or isotype
specific secondary antibodies or by specifically labelling the
control antibody with a detectable label), the method can be used
to determine that the test antibody reduces the binding of the
control antibody to the COV-S antigen, indicating that the test
antibody recognizes substantially the same epitope as the control
antibody (e.g., antibodies as described herein and in FIGS. 1, 2
and 36). The binding of the (labeled) control antibody in the
presence of a completely irrelevant antibody (that does not bind
CoV-S) can serve as the control high value. The control low value
can be obtained by incubating the labeled control antibody with the
same but unlabeled control antibody, where competition would occur
and reduce binding of the labeled antibody. In a test assay, a
significant reduction in labeled antibody reactivity in the
presence of a test antibody is indicative of a test antibody that
recognizes substantially the same epitope, i.e., one that competes
with the labeled control antibody. For example, any test antibody
that reduces the binding of any one of antibodies described herein
and in FIGS. 1, 2 and 36, to both of SARS-CoV-S or SARS-CoV-2-S
antigens by at least about 50%, such as at least about 60%, or more
preferably at least about 70% (e.g., about 65-100%), at any ratio
of control antibody as described herein and in FIGS. 1, 2 and 36,
test antibody between about 1:1 or 1:10 and about 1:100 is
considered to be an antibody that binds to substantially the same
epitope or determinant as any one of antibodies described herein
and in FIGS. 1, 2 and 36, respectively. Preferably, such test
antibody will reduce the binding of any one of antibodies as
described herein and in FIGS. 1, 2 and 36, to at least one,
preferably each, of the SARS-CoV-S or SARS-CoV-2-S antigens
preferably at least about 50%, at least about 60%, at least about
80% or at least about 90% (e.g., about 95%) of the binding of any
one of antibodies described herein and in FIGS. 1, 2 and 36,
observed in the absence of the test antibody. These methods can be
adapted to identify and/or evaluate antibodies that compete with
other control antibodies.
[0365] A simple competition assay in which a test antibody is
applied at saturating concentration to a surface onto which either
SARS-CoV-S or SARS-CoV-2-S, or both, are immobilized also may be
advantageously employed. The surface in the simple competition
assay is preferably of a media suitable for OCTET.RTM. and/or
PROTEON.RTM.. The binding of a control antibody (e.g., any one of
antibodies described herein and in FIGS. 1, 2 and 36) to the
CoV-S-coated surface is measured. This binding to the
CoV-S-containing surface of the control antibody alone is compared
with the binding of the control antibody in the presence of a test
antibody. A significant reduction in binding to the
CoV-S-containing surface by the control antibody in the presence of
a test antibody indicates that the test antibody recognizes
substantially the same epitope as the control antibody such that
the test antibody "competes" with the control antibody. Any test
antibody that reduces the binding of control antibody (such as
anyone of antibodies described herein and in FIGS. 1, 2 and 36) to
both of SARS-CoV-S and SARS-CoV-2-S antigens by at least about 20%
or more, at least about 40%, at least about 50%, at least about
70%, or more, can be considered to be an antibody that binds to
substantially the same epitope or determinant as the control
antibody (e.g., any one of antibodies described herein and in FIGS.
1, 2 and 36). Preferably, such test antibody will reduce the
binding of the control antibody (e.g., any one of antibodies
described herein and in FIGS. 1, 2 and 36) to the CoV-S antigen by
at least about 50% (e.g., at least about 60%, at least about 70%,
or more). It will be appreciated that the order of control and test
antibodies can be reversed; i.e. the control antibody can be first
bound to the surface and then the test antibody is brought into
contact with the surface thereafter in a competition assay.
Preferably, the antibody having higher affinity for SARS-CoV-S and
SARS-CoV-2-S is bound to the CoV-S-containing surface first, as it
will be expected that the decrease in binding seen for the second
antibody (assuming the antibodies are competing) will be of greater
magnitude. Further examples of such assays are provided in, e.g.,
Saunal and Regenmortel, J. Immunol. Methods, 183:33-41 (1989), the
disclosure of which is incorporated herein by reference.
[0366] Determination of whether an antibody, antigen-binding
fragment thereof, or antibody derivative, e.g., an affinity-matured
antibody or antigen binding fragment of any of the anti-CoV-S
antibodies exemplified herein, binds within one of the epitope
regions defined above can be carried out in ways known to the
person skilled in the art. In another example of such
mapping/characterization methods, an epitope region for an
anti-CoV-S antibody may be determined by epitope "footprinting"
using chemical modification of the exposed amines/carboxyls in the
SARS-CoV-S and SARS-CoV-2-S protein. One specific example of such a
foot-printing technique is the use of hydrogen-deuterium exchange
detected by mass spectrometry ("HXMS"), wherein a
hydrogen/deuterium exchange of receptor and ligand protein amide
protons, binding, and back exchange occurs, wherein the backbone
amide groups participating in protein binding are protected from
back exchange and therefore will remain deuterated. Relevant
regions can be identified at this point by peptic proteolysis, fast
microbore high-performance liquid chromatography separation, and/or
electrospray ionization mass spectrometry (See, e.g., Ehring H.,
Analytical Biochemistry, 267(2):252-259 (1999) and Engen, J. R.
& Smith, D. L., Anal. Chem., 73:256A-265A (2001)). Another
example of a suitable epitope identification technique is nuclear
magnetic resonance epitope mapping ("NMR"), where typically the
position of the signals in two-dimensional NMR spectras of the free
antigen and the antigen complexed with the antigen binding peptide,
such as an antibody, are compared. The antigen typically is
selectively isotopically labeled with .sup.15N so that only signals
corresponding to the antigen and no signals from the antigen
binding peptide are seen in the NMR-spectrum. Antigen signals
originating from amino acids involved in the interaction with the
antigen binding peptide typically will shift position in the
spectras of the complex compared to the spectras of the free
antigen, and the amino acids involved in the binding can be
identified that way. See, e.g., Ernst Schering Res. Found.
Workshop, (44):149-67 (2004); Huang et al., J. Mol. Biol.,
281(1):61-67 (1998); and Saito and Patterson, Methods, 9(3):516-24
(1996). Epitope mapping/characterization also can be performed
using mass spectrometry ("MS") methods (See, e.g., Downard, J. Mass
Spectrom., 35(4):493-503 (2000) and Kiselar and Downard, Anal.
Chem., 71(9):1792-801 (1999)).
[0367] Protease digestion techniques also can be useful in the
context of epitope mapping and identification. Antigenic
determinant-relevant regions/sequences can be determined by
protease digestion, e.g. by using trypsin in a ratio of about 1:50
to SARS-CoV-S or SARS-CoV-2-S overnight ("o/n") digestion at
37.degree. C. and pH 7-8, followed by mass spectrometry ("MS")
analysis for peptide identification. The peptides protected from
trypsin cleavage by the anti-CoV-S antibody can subsequently be
identified by comparison of samples subjected to trypsin digestion
and samples incubated with antibody and then subjected to digestion
by e.g. trypsin (thereby revealing a footprint for the antibody).
Other enzymes like chymotrypsin or pepsin can be used in similar
epitope characterization methods. Moreover, enzymatic digestion can
provide a quick method for analyzing whether a potential antigenic
determinant sequence is within a region of CoV-S in the context of
a CoV-S-binding polypeptide. If the polypeptide is not surface
exposed, it is most likely not relevant in terms of
immunogenicity/antigenicity (See, e.g., Manca, Ann. Ist. Super.
Sanita., 27(1):15-9 (1991) for a discussion of similar
techniques).
[0368] Site-directed mutagenesis is another technique useful for
characterization of a binding epitope. For example, in
"alanine-scanning" site-directed mutagenesis (also known as alanine
scanning, alanine scanning mutagenesis, alanine scanning mutations,
combinatorial alanine scanning, or creation of alanine point
mutations, for example), each residue within a protein segment is
replaced with an alanine residue (or another residue such as valine
where alanine is present in the wild-type sequence) through such
methodologies as direct peptide or protein synthesis, site-directed
mutagenesis, the GENEART.TM. Mutagenesis Service (Thermo Fisher
Scientific, Waltham, Mass. U.S.A.) or shotgun mutagenesis, for
example. A series of single point mutants of the molecule is
thereby generated using this technique; the number of mutants
generated is equivalent to the number of residues in the molecule,
each residue being replaced, one at a time, by a single alanine
residue. Alanine is generally used to replace native (wild-type)
residues because of its non-bulky, chemically inert, methyl
functional group that can mimic the secondary structure preferences
that many other amino acids may possess. Subsequently, the effects
replacing a native residue with an alanine has on binding affinity
of an alanine scanning mutant and its binding partner can be
measured using such methods as, but not limited to, SPR binding
experiments. If a mutation leads to a significant reduction in
binding affinity, it is most likely that the mutated residue is
involved in binding. Monoclonal antibodies specific for structural
epitopes (i.e., antibodies that do not bind the unfolded protein)
can be used as a positive control for binding affinity experiments
to verify that the alanine-replacement does not influence the
overall tertiary structure of the protein (as changes to the
overall fold of the protein may indirectly affect binding and
thereby produce a false positive result). See, e.g., Clackson and
Wells, Science, 267:383-386 (1995); Weiss et al., Proc. Natl. Acad.
Sci. USA, 97(16):8950-8954 (2000); and Wells, Proc. Natl. Acad.
Sci. USA, 93:1-6 (1996). Example 5 identifies the specific epitope
or residues of CoV-S which specifically interact with the
anti-CoV-S antibodies disclosed herein.
[0369] Electron microscopy can also be used for epitope
"footprinting". For example, Wang et al., Nature, 355:275-278
(1992) used coordinated application of cryoelectron microscopy,
three-dimensional image reconstruction, and X-ray crystallography
to determine the physical footprint of a Fab-fragment on the capsid
surface of native cowpea mosaic virus.
[0370] Other forms of "label-free" assay for epitope evaluation
include SPR (sold commercially as the BIACORE.RTM. system, GE
Healthcare Life Sciences, Marlborough, Mass.) and reflectometric
interference spectroscopy ("RifS") (See, e.g., Fagerstam et al.,
Journal of Molecular Recognition, 3:208-14 (1990); Nice et al., J.
Chromatogr., 646:159-168 (1993); Leipert et al., Angew. Chem. Int.
Ed., 37:3308-3311 (1998); Kroger et al., Biosensors and
Bioelectronics, 17:937-944 (2002)).
[0371] The expressions "framework region" or "FR" refer to one or
more of the framework regions within the variable regions of the
light and heavy chains of an antibody (See Kabat et al., Sequences
of Proteins of Immunological Interest, 4.sup.th edition, Bethesda,
Md.: U.S. Dept. of Health and Human Services, Public Health
Service, National Institutes of Health (1987)). These expressions
include those amino acid sequence regions interposed between the
CDRs within the variable regions of the light and heavy chains of
an antibody.
[0372] The term "Fc region" is used to define a C-terminal region
of an immunoglobulin heavy chain. The "Fc region" may be a native
sequence Fc region or a variant Fc region. Although the boundaries
of the Fc region of an immunoglobulin heavy chain might vary, the
human IgG heavy chain Fc region is usually defined to stretch from
an amino acid residue at position Cys226, or from Pro230, to the
carboxyl-terminus thereof. The numbering of the residues in the Fc
region is that of the EU index as in Kabat. Kabat et al., Sequences
of Proteins of Immunological Interest, 5th edition, Bethesda, Md.:
U.S. Dept. of Health and Human Services, Public Health Service,
National Institutes of Health (1991). The Fc region of an
immunoglobulin generally comprises two constant domains, CH2 and
CH3.
[0373] The terms "Fc receptor" and "FcR" describe a receptor that
binds to the Fc region of an antibody. The preferred FcR is a
native sequence human FcR. Moreover, a preferred FcR is one that
binds an IgG antibody (a gamma receptor) and includes receptors of
the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses,
including allelic variants and alternatively spliced forms of these
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. FcRs are reviewed in
Ravetch and Kinet, Ann. Rev. Immunol., 9:457-92 (1991); Capel et
al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med., 126:330-41 (1995). "FcR" also includes the neonatal
receptor, FcRn, which is responsible for the transfer of maternal
IgGs to the fetus (Guyer et al., J. Immunol., 117:587 (1976); and
Kim et al., J. Immunol., 24:249 (1994)), and which primarily
functions to modulate and/or extend the half-life of antibodies in
circulation. To the extent that the disclosed anti-CoV-S antibodies
are aglycosylated, as a result of the expression system and/or
sequence, the subject antibodies are expected to bind FcRn
receptors, but not to bind (or to minimally bind) Fc.gamma.
receptors.
[0374] A "functional Fc region" possesses at least one effector
function of a native sequence Fc region. Exemplary "effector
functions" include C1q binding; complement dependent cytotoxicity
("CDC"); Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity ("ADCC"); phagocytosis; down-regulation of cell
surface receptors (e.g. B cell receptor ("BCR")), etc. Such
effector functions generally require the Fc region to be combined
with a binding domain (e.g. an antibody variable domain) and can be
assessed using various assays known in the art for evaluating such
antibody effector functions.
[0375] A "native sequence Fc region" comprises an amino acid
sequence identical to the amino acid sequence of an Fc region found
in nature. A "variant Fc region" comprises an amino acid sequence
that differs from that of a native sequence Fc region by virtue of
at least one amino acid modification, yet retains at least one
effector function of the native sequence Fc region. Preferably, the
variant Fc region has at least one amino acid substitution compared
to a native sequence Fc region or to the Fc region of a parent
polypeptide, e.g. from about one to about ten amino acid
substitutions, and preferably from about one to about five amino
acid substitutions in a native sequence Fc region or in the Fc
region of the parent polypeptide. The variant Fc region herein will
preferably possess at least about 80% sequence identity with a
native sequence Fc region and/or with an Fc region of a parent
polypeptide, and most preferably at least about 90% sequence
identity therewith, more preferably at least about 95%, at least
about 96%, at least about 97%, at least about 98%, or at least
about 99% sequence identity therewith.
[0376] In some embodiments, the Fc region of an antibody or
antigen-binding antibody fragment of the present disclosure may
bind to an Fc receptor (FcR). The FcR may be, but is not limited
to, Fc gamma receptor (FcgR), FcgRI, FcgRIIA, FcgRIIB1, FcgRIIB2,
FcgRIIIA, FcgRIIIB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc
alpha receptor (FcaR), FcaRI, Fc alpha/mu receptor (Fca/mR), or
neonatal Fc receptor (FcRn). The Fc may be an IgM, IgD, IgG, IgE,
or IgA isotype. An IgG isotype may be an IgG1, IgG2, IgG3, or
IgG4.
[0377] Certain amino acid modifications in the Fc region are known
to modulate Ab effector functions and properties, such as, but not
limited to, antibody-dependent cellular cytotoxicity (ADCC),
antibody-dependent cellular phagocytosis (ADCP), complement
dependent cytotoxicity (CDC), and half-life (Wang X. et al.,
Protein Cell. 2018 January; 9(1): 63-73; Dall'Acqua W. F. et al., J
Biol Chem. 2006 Aug. 18; 281(33):23514-24. Epub 2006 Jun. 21;
Monnet C. et al, Front Immunol. 2015 Feb. 4; 6:39. doi:
10.3389/fimmu.2015.00039. eCollection 2015). The mutation may be
symmetrical or asymmetrical. In certain cases, antibodies with Fc
regions that have asymmetrical mutation(s) (i.e., two Fc regions
are not identical) may provide better functions such as ADCC (Liu
Z. et al. J Biol Chem. 2014 Feb. 7; 289(6): 3571-3590).
[0378] Any of the antibody variable region sequences disclosed
herein may be used in combination with a wild-type (WT) Fc or a
variant Fc. In particular embodiments, an Fc selected from the Fc
sequences described in Table 1 may be used. Any of the variable
region sequences disclosed herein may be used in combination with
any appropriate Fc including any of the Fc variants provided in
Table 1 to form an antibody or an antigen-binding antibody fragment
of the present disclosure. The lysine (K) at the C-terminus of each
Fc may be present or absent.
TABLE-US-00001 TABLE 1 Exemplary Fc Variants. Fc Variant Name SEQ
ID NO: Differences from WT WT 11 0 YTE 12 3 LA 13 2 LS 14 2 LA-RE
15 4 DEL 16 2 LALA-DEL 17 4
[0379] An IgG1-type Fc optionally may comprise one or more amino
acid substitutions. Such substitutions may include, for example,
N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P,
L234V, G236-deleted, P238A, A327Q, A327G, P329A, K322A, L234F,
L235E, P331S, T394D, A330L, P331S, F243L, R292P, Y300L, V3051,
P396L, S239D, 1332E, S298A, E333A, K334A, L234Y, L235Q, G236W,
S239M, H268D, D270E, K326D, A330M, K334E, G236A, K326W, S239D,
E333S, S267E, H268F, S324T, E345R, E430G, S440Y, M428L, N434S,
L328F, M252Y, S254T, T256E, and/or any combination thereof (the
residue numbering is according to the EU index as in Kabat)
(Dall'Acqua W. F. et al., J Biol Chem. 2006 Aug. 18;
281(33):23514-24. Epub 2006 Jun. 21; Wang X. et al., Protein Cell.
2018 January; 9(1): 63-73), or for example, N434A, Q438R, S440E,
L432D, N434L, and/or any combination thereof (the residue numbering
according to EU numbering). The Fc region may further comprise one
or more additional amino acid substitutions. Such substitutions may
include but are not limited to A330L, L234F, L235E, P3318, and/or
any combination thereof (the residue numbering is according to the
EU index as in Kabat). Specific exemplary substitution combinations
for an IgG1-type Fc include, but not limited to: M252Y, S254T, and
T256E ("YTE" variant); M428L and N434A ("LA" variant), M428L and
N434S ("LS" variant); M428L, N434A, Q438R, and S440E ("LA-RE"
variant); L432D and N434L ("DEL" variant); and L234A, L235A, L432D,
and N434L ("LALA-DEL" variant) (the residue numbering is according
to the EU index as in Kabat). In particular embodiments, an
IgG1-type Fc variant may comprise the amino acid sequence of SEQ ID
NOS: 11, 12, 13, 14, 15, 16, or 17. In one embodiment, the Fc
variant is an LA variant and comprises the amino acid sequence of
SEQ ID NO: 13.
[0380] When the Ab is an IgG2, the Fc region optionally may
comprise one or more amino acid substitutions. Such substitutions
may include but are not limited to P238S, V234A, G237A, H268A,
H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S,
M252Y, S254T, T256E, and/or any combination thereof (the residue
numbering is according to the EU index as in Kabat). The Fc region
optionally may further comprise one or more additional amino acid
substitutions. Such substitutions may include but are not limited
to M252Y, S254T, T256E, and/or any combination thereof (the residue
numbering is according to the EU index as in Kabat).
[0381] An IgG3-type Fc region optionally may comprise one or more
amino acid substitutions. Such substitutions may include but are
not limited to E235Y (the residue numbering is according to the EU
index as in Kabat).
[0382] An IgG4-type Fc region optionally may comprise one or more
amino acid substitutions. Such substitutions may include but are
not limited to, E233P, F234V, L235A, G237A, E318A, S228P, L236E,
S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and/or any
combination thereof (the residue numbering is according to the EU
index as in Kabat). The substitution may be, for example, S228P
(the residue numbering is according to the EU index as in
Kabat).
[0383] In some cases, the glycan of the human-like Fc region may be
engineered to modify the effector function (for example, see Li T.
et al., Proc Natl Acad Sci USA. 2017 Mar. 28; 114(13):3485-3490.
doi: 10.1073/pnas.1702173114. Epub 2017 Mar. 13).
[0384] An "isolated" antibody, as used herein, refers to an
antibody that is substantially free of other antibodies having
different antigenic specificities. In some embodiments, an isolated
antibody is substantially free of other unintended cellular
material and/or chemicals.
[0385] As used herein, "specific binding" or "specifically binds"
means that the interaction of the antibody, or antigen-binding
portion thereof, with an antigen is dependent upon the presence of
a particular structure (e.g., antigenic determinant or epitope).
For example, the antibody, or antigen-binding portion thereof,
binds to a specific protein, rather than proteins generally. In
some embodiments, an antibody, or antigen-binding portion thereof,
specifically binds a target, e.g., SARS-CoV-S and/or SARS-CoV-S-2.
In some embodiments, an antibody, or antigen-binding portion
thereof, specifically binds to more than one coronavirus spike
protein, e.g., the spike protein of SARS-CoV-S and the spike
protein of SARS-CoV-2-S, for example. In some embodiments, the
antibody, or antigen-binding portion thereof, specifically binds to
two different, but related, antigens, e.g., the spike protein of
SARS-CoV1-S and the spike protein of SARS-CoV2-S, e.g., via a
conserved epitope.
B. Anti-CoV-S Antibodies and Binding Fragments Thereof Having
Binding Activity for CoV-S
[0386] CoV-S refers to the S protein of a coronavirus which is
expressed on the surface of virions as a structural protein. As
mentioned previously, the S protein plays an essential role for
coronaviruses in binding to receptors on the host cell and
determines host tropism (Zhu Z. et al., Infect Genet Evol. 2018
July; 61:183-184). SARS-CoV and SARS-CoV-2 bind to
angiotensin-converting enzyme 2 (ACE2) of the host cell via the S
protein's receptor-binding domains (RBDs) and uses ACE2 as a
receptor to enter the host cells (Ge X. Y. et al., Nature. 2013
Nov. 28; 503(7477):535-8. doi: 10.1038/nature12711. Epub 2013 Oct.
30.; Hoffmann M. et al., Cell. 2020 Mar. 4. pii:
S0092-8674(20)30229-4). SARS-CoV can also use CD209L (also known as
L-SIGN) as an alternative receptor (Jeffers S. A. et al., Proc Natl
Acad Sci USA. 2004 Nov. 2; 101(44):15748-53. Epub 2004 Oct. 20).
MERS-CoV binds dipeptidyl peptidase 4 ("DPP4", also known as CD26)
of the host cells via a different RBD of the S protein. Cell entry
of coronaviruses depends on not only binding of the S protein to a
host cell receptor but often also priming of the S protein by host
cell proteases, and recently SARS-CoV-2 was found to use the serine
protease TMPRSS2 for S protein priming and then ACE2 for entry (Wu
A. et al., Cell Host Microbe. 2020 Mar. 11; 27(3):325-328; Hoffmann
M. et al., Cell. 2020 Mar. 4. pii: S0092-8674(20)30229-4).
[0387] The S protein of SARS-CoV is referred to as SARS-CoV-S and
may for example comprise the amino acid sequence of SEQ ID NO: 1
(1288 amino acids). The S protein of SARS-CoV-2 is referred to as
SARS-CoV-2-S and may for example comprise the amino acid sequence
of SEQ ID NO: 5 (1273 amino acids).
[0388] The present disclosure provides exemplary antibodies and
antigen-binding antibody fragments that specifically bind to CoV,
wherein at least some of these antibodies and antigen-binding
antibody fragments specifically bind to SARS-CoV-2-S and/or
SARS-CoV-2-S. Due to the sequence similarity among different CoV
species, such antibodies or antigen-binding antibody fragments of
the present disclosure may also cross react with the S protein of
other CoV species.
[0389] The exemplary S proteins of CoV that the antibodies or
antigen-binding antibody fragments of the present disclosure may
specifically bind include by way of example, Bat SARS CoV (GenBank
Accession No. FJ211859), SARS CoV (GenBank Accession No. FJ211860),
BtSARS.HKU3.1 (GenBank Accession No. DQ022305), BtSARS.HKU3.2
(GenBank Accession No. DQ084199), BtSARS.HKU3.3 (GenBank Accession
No. DQ084200), BtSARS.Rm1 (GenBank Accession No. DQ412043),
BtCoV.279.2005 (GenBank Accession No. DQ648857), BtSARS.Rf1
(GenBank Accession No. DQ412042), BtCoV.273.2005 (GenBank Accession
No. DQ648856), BtSARS.Rp3 (GenBank Accession No. DQ071615), SARS
CoV.A022 (GenBank Accession No. AY686863), SARSCoV.CUHK-W1 (GenBank
Accession No. AY278554), SARSCoV.GDO1 (GenBank Accession No.
AY278489), SARSCoV.HC.SZ.61.03 (GenBank Accession No. AY515512),
SARSCoV.SZ16 (GenBank Accession No. AY304488), SARSCoV.Urbani
(GenBank Accession No. AY278741), SARSCoV.civet010 (GenBank
Accession No. AY572035), or SARSCoV.MA.15 (GenBank Accession No.
DQ497008), Rs SHC014 (GenBank.RTM. Accession No. KC881005), Rs3367
(GenBank.RTM. Accession No. KC881006), WiV1 S (GenBank.RTM.
Accession No. KC881007).
[0390] In some embodiments, the antibodies and antigen-binding
antibody fragments provided herein may also bind to and neutralize
existing bat CoV or pre-emergent bat CoVs. Antibodies and
antigen-binding antibody fragments with such binding and/or
neutralization abilities would be particularly useful in a future
pandemic that may be caused by a spillover from an animal
reservoir, like a bat. In fact, ADI-55688, ADI-55689, ADI-55993,
ADI-5600, ADI-56046, ADI-55690, ADI-56010, and ADI-55951 were shown
to neutralize authentic bat coronavirus, WIV1 (see FIG. 3E of Wec
A. et al., Science. 2020 Jun. 15; eabc7424. doi:
10.1126/science.abc7424).
[0391] Alternatively, the S proteins of CoV to which the antibodies
or antigen-binding antibody fragments of the present disclosure may
specifically bind to and neutralize pre-emergent coronaviruses from
other species, e.g., bats.
[0392] Still alternatively, the S proteins of CoV to which the
antibodies or antigen-binding antibody fragments of the present
disclosure may specifically bind to may include, for example,
Middle East respiratory syndrome coronavirus isolate Riyadh_2_2012
(GenBank Accession No. KF600652.1), Middle East respiratory
syndrome coronavirus isolate Al-Hasa_18_2013 (GenBank Accession No.
KF600651.1), Middle East respiratory syndrome coronavirus isolate
Al-Hasa_17_2013 (GenBank Accession No. KF600647.1), Middle East
respiratory syndrome coronavirus isolate Al-Hasa_15_2013 (GenBank
Accession No. KF600645.1), Middle East respiratory syndrome
coronavirus isolate Al-Hasa_16_2013 (GenBank Accession No.
KF600644.1), Middle East respiratory syndrome coronavirus isolate
Al-Hasa_21_2013 (GenBank Accession No. KF600634), Middle East
respiratory syndrome coronavirus isolate Al-Hasa_19_2013 (GenBank
Accession No. KF600632), Middle East respiratory syndrome
coronavirus isolate Buraidah_1_2013 (GenBank Accession No.
KF600630.1), Middle East respiratory syndrome coronavirus isolate
Hafr-Al-Batin_1_2013 (GenBank Accession No. KF600628.1), Middle
East respiratory syndrome coronavirus isolate Al-Hasa_12_2013
(GenBank Accession No. KF600627.1), Middle East respiratory
syndrome coronavirus isolate Bisha_1_2012 (GenBank Accession No.
KF600620.1), Middle East respiratory syndrome coronavirus isolate
Riyadh_3_2013 (GenBank Accession No. KF600613.1), Middle East
respiratory syndrome coronavirus isolate Riyadh_1_2012 (GenBank
Accession No. KF600612.1), Middle East respiratory syndrome
coronavirus isolate Al-Hasa_3_2013 (GenBank Accession No.
KF186565.1), Middle East respiratory syndrome coronavirus isolate
Al-Hasa_1_2013 (GenBank Accession No. KF186567.1), Middle East
respiratory syndrome coronavirus isolate Al-Hasa_2_2013 (GenBank
Accession No. KF186566.1), Middle East respiratory syndrome
coronavirus isolate Al-Hasa_4_2013 (GenBank Accession No.
KF186564.1), Middle East respiratory syndrome coronavirus (GenBank
Accession No. KF192507.1), Betacoronavirus England 1-N1 (GenBank
Accession No. NC_019843), MERS-CoV_SA-N1 (GenBank Accession No.
KC667074), following isolates of Middle East Respiratory Syndrome
Coronavirus (GenBank Accession No: KF600656.1, GenBank Accession
No: KF600655.1, GenBank Accession No: KF600654.1, GenBank Accession
No: KF600649.1, GenBank Accession No: KF600648.1, GenBank Accession
No: KF600646.1, GenBank Accession No: KF600643.1, GenBank Accession
No: KF600642.1, GenBank Accession No: KF600640.1, GenBank Accession
No: KF600639.1, GenBank Accession No: KF600638.1, GenBank Accession
No: KF600637.1, GenBank Accession No: KF600636.1, GenBank Accession
No: KF600635.1, GenBank Accession No: KF600631.1, GenBank Accession
No: KF600626.1, GenBank Accession No: KF600625.1, GenBank Accession
No: KF600624.1, GenBank Accession No: KF600623.1, GenBank Accession
No: KF600622.1, GenBank Accession No: KF600621.1, GenBank Accession
No: KF600619.1, GenBank Accession No: KF600618.1, GenBank Accession
No: KF600616.1, GenBank Accession No: KF600615.1, GenBank Accession
No: KF600614.1, GenBank Accession No: KF600641.1, GenBank Accession
No: KF600633.1, GenBank Accession No: KF600629.1, GenBank Accession
No: KF600617.1), Coronavirus Neoromicia/PML-PHE1/RSA/2011 GenBank
Accession: KC869678.2, Bat Coronavirus
Taper/CII_KSA_287/Bisha/Saudi Arabia/GenBank Accession No:
KF493885.1, Bat coronavirus Rhhar/CII_KSA_003/Bisha/Saudi
Arabia/2013 GenBank Accession No: KF493888.1, Bat coronavirus
Pikuh/CII_KSA_001/Riyadh/Saudi Arabia/2013 GenBank Accession No:
KF493887.1, Bat coronavirus Rhhar/CII_KSA_002/Bisha/Saudi
Arabia/2013 GenBank Accession No: KF493886.1, Bat Coronavirus
Rhhar/CII_KSA_004/Bisha/Saudi Arabia/2013 GenBank Accession No:
KF493884.1, BtCoV.HKU4.2 (GenBank Accession No. EF065506),
BtCoV.HKU4.1 (GenBank Accession No. NC_009019), BtCoV.HKU4.3
(GenBank Accession No. EF065507), BtCoV.HKU4.4 (GenBank Accession
No. EF065508), BtCoV 133.2005 (GenBank Accession No. NC 008315),
BtCoV.HKU5.5 (GenBank Accession No. EF065512); BtCoV.HKU5.1
(GenBank Accession No. NC_009020), BtCoV.HKU5.2 (GenBank Accession
No. EF065510), BtCoV.HKU5.3 (GenBank Accession No. EF065511), human
betacoronavirus 2c Jordan-N3/2012 (GenBank Accession No.
KC776174.1; human betacoronavirus 2c EMC/2012 (GenBank Accession
No. JX869059.2), Pipistrellus bat coronavirus HKU5 isolates
(GenBank Accession No:KC522089.1, GenBank Accession No:KC522088.1,
GenBank Accession No:KC522087.1, GenBank Accession No:KC522086.1,
GenBank Accession No:KC522085.1, GenBank Accession No: KC522084.1,
GenBank Accession No:KC522083.1, GenBank Accession No:KC522082.1,
GenBank Accession No:KC522081.1, GenBank Accession No:KC522080.1,
GenBank Accession No:KC522079.1, GenBank Accession No:KC522078.1,
GenBank Accession No: KC522077.1, GenBank Accession No:KC522076.1,
GenBank Accession No:KC522075.1, GenBank Accession No:KC522104.1,
GenBank Accession No:KC522104.1, GenBank Accession No:KC522103.1,
GenBank Accession No:KC522102.1, GenBank Accession No: KC522101.1,
GenBank Accession No:KC522100.1, GenBank Accession No:KC522099.1,
GenBank Accession No:KC522098.1, GenBank Accession No:KC522097.1,
GenBank Accession No:KC522096.1, GenBank Accession No:KC522095.1,
GenBank Accession No: KC522094.1, GenBank Accession No:KC522093.1,
GenBank Accession No:KC522092.1, GenBank Accession No:KC522091.1,
GenBank Accession No:KC522090.1, GenBank Accession No:KC522119.1
GenBank Accession No:KC522118.1 GenBank Accession No: KC522117.1
GenBank Accession No:KC522116.1 GenBank Accession No:KC522115.1
GenBank Accession No:KC522114.1 GenBank Accession No:KC522113.1
GenBank Accession No:KC522112.1 GenBank Accession No:KC522111.1
GenBank Accession No: KC522110.1 GenBank Accession No:KC522109.1
GenBank Accession No:KC522108.1, GenBank Accession No:KC522107.1,
GenBank Accession No:KC522106.1, GenBank Accession No:KC522105.1)
Pipistrellus bat coronavirus HKU4 isolates (GenBank Accession
No:KC522048.1, GenBank Accession No:KC522047.1, GenBank Accession
No:KC522046.1, GenBank Accession No:KC522045.1, GenBank Accession
No: KC522044.1, GenBank Accession No:KC522043.1, GenBank Accession
No:KC522042.1, GenBank Accession No:KC522041.1, GenBank Accession
No:KC522040.1 GenBank Accession No:KC522039.1, GenBank Accession
No:KC522038.1, GenBank Accession No:KC522037.1, GenBank Accession
No:KC522036.1, GenBank Accession No:KC522048.1 GenBank Accession
No:KC522047.1 GenBank Accession No:KC522046.1 GenBank Accession
No:KC522045.1 GenBank Accession No:KC522044.1 GenBank Accession
No:KC522043.1 GenBank Accession No:KC522042.1 GenBank Accession
No:KC522041.1 GenBank Accession No:KC522040.1, GenBank Accession
No:KC522039.1 GenBank Accession No:KC522038.1 GenBank Accession
No:KC522037.1 GenBank Accession No:KC522036.1, GenBank Accession
No:KC522061.1 GenBank Accession No:KC522060.1 GenBank Accession
No:KC522059.1 GenBank Accession No:KC522058.1 GenBank Accession
No:KC522057.1 GenBank Accession No:KC522056.1 GenBank Accession
No:KC522055.1 GenBank Accession No:KC522054.1 GenBank Accession
No:KC522053.1 GenBank Accession No:KC522052.1 GenBank Accession
No:KC522051.1 GenBank Accession No:KC522050.1 GenBank Accession
No:KC522049.1 GenBank Accession No:KC522074.1, GenBank Accession
No:KC522073.1 GenBank Accession No:KC522072.1 GenBank Accession
No:KC522071.1 GenBank Accession No:KC522070.1 GenBank Accession
No:KC522069.1 GenBank Accession No:KC522068.1 GenBank Accession
No:KC522067.1, GenBank Accession No:KC522066.1 GenBank Accession
No:KC522065.1 GenBank Accession No:KC522064.1, GenBank Accession
No:KC522063.1, or GenBank Accession No:KC522062.1.
[0393] Alternatively, the S proteins of CoV to which the antibodies
or antigen-binding antibody fragments of the present disclosure may
specifically bind may include for example,
FCov.FIPV.79.1146.VR.2202 (GenBank Accession No. NV_007025),
transmissible gastroenteritis virus (TGEV) (GenBank Accession No.
NC_002306; GenBank Accession No. Q811789.2; GenBank Accession No.
DQ811786.2; GenBank Accession No. DQ811788.1; GenBank Accession No.
DQ811785.1; GenBank Accession No. X52157.1; GenBank Accession No.
AJ011482.1; GenBank Accession No. KC962433.1; GenBank Accession No.
AJ271965.2; GenBank Accession No. JQ693060.1; GenBank Accession No.
KC609371.1; GenBank Accession No. JQ693060.1; GenBank Accession No.
JQ693059.1; GenBank Accession No. JQ693058.1; GenBank Accession No.
JQ693057.1; GenBank Accession No. JQ693052.1; GenBank Accession No.
JQ693051.1; GenBank Accession No. JQ693050.1), or porcine
reproductive and respiratory syndrome virus (PRRSV) (GenBank
Accession No. NC_001961.1; GenBank Accession No. DQ811787).
[0394] Alternatively, the S proteins of CoV to which the antibodies
or antigen-binding antibody fragments of the present disclosure may
specifically bind may include, for example, BtCoV.1A.AFCD62
(GenBank Accession No. NC_010437), BtCoV.1B.AFCD307 (GenBank
Accession No. NC_010436), BtCov.HKU8.AFCD77 (GenBank Accession No.
NC_010438), BtCoV.512.2005 (GenBank Accession No. DQ648858),
porcine epidemic diarrhea virus PEDV.CV777 (GenBank Accession No.
NC_003436, GenBank Accession No. DQ355224.1, GenBank Accession No.
DQ355223.1, GenBank Accession No. DQ355221.1, GenBank Accession No.
JN601062.1, GenBank Accession No. N601061.1, GenBank Accession No.
JN601060.1, GenBank Accession No. JN601059.1, GenBank Accession No.
JN601058.1, GenBank Accession No. JN601057.1, GenBank Accession No.
JN601056.1, GenBank Accession No. JN601055.1, GenBank Accession No.
JN601054.1, GenBank Accession No. JN601053.1, GenBank Accession No.
JN601052.1, GenBank Accession No. JN400902.1, GenBank Accession No.
JN547395.1, GenBank Accession No. FJ687473.1, GenBank Accession No.
FJ687472.1, GenBank Accession No. FJ687471.1, GenBank Accession No.
FJ687470.1, GenBank Accession No. FJ687469.1, GenBank Accession No.
FJ687468.1, GenBank Accession No. FJ687467.1, GenBank Accession No.
FJ687466.1, GenBank Accession No. FJ687465.1, GenBank Accession No.
FJ687464.1, GenBank Accession No. FJ687463.1, GenBank Accession No.
FJ687462.1, GenBank Accession No. FJ687461.1, GenBank Accession No.
FJ687460.1, GenBank Accession No. FJ687459.1, GenBank Accession No.
FJ687458.1, GenBank Accession No. FJ687457.1, GenBank Accession No.
FJ687456.1, GenBank Accession No. FJ687455.1, GenBank Accession No.
FJ687454.1, GenBank Accession No. FJ687453 GenBank Accession No.
FJ687452.1, GenBank Accession No. FJ687451.1, GenBank Accession No.
FJ687450.1, GenBank Accession No. FJ687449.1, GenBank Accession No.
AF500215.1, GenBank Accession No. KF476061.1, GenBank Accession No.
KF476060.1, GenBank Accession No. KF476059.1, GenBank Accession No.
KF476058.1, GenBank Accession No. KF476057.1, GenBank Accession No.
KF476056.1, GenBank Accession No. KF476055.1, GenBank Accession No.
KF476054.1, GenBank Accession No. KF476053.1, GenBank Accession No.
KF476052.1, GenBank Accession No. KF476051.1, GenBank Accession No.
KF476050.1, GenBank Accession No. KF476049.1, GenBank Accession No.
KF476048.1, GenBank Accession No. KF177258.1, GenBank Accession No.
KF177257.1, GenBank Accession No. KF177256.1, GenBank Accession No.
KF177255.1), HCoV.229E (GenBank Accession No. NC_002645),
HCoV.NL63.Amsterdam.I (GenBank Accession No. NC_005831),
BtCoV.HKU2.HK.298.2006 (GenBank Accession No. EF203066),
BtCoV.HKU2.HK.33.2006 (GenBank Accession No. EF203067),
BtCoV.HKU2.HK.46.2006 (GenBank Accession No. EF203065), or
BtCoV.HKU2.GD.430.2006 (GenBank Accession No. EF203064).
[0395] Alternatively, the S proteins of CoV to which the antibodies
or antigen-binding antibody fragments of the present disclosure may
specifically bind may include, for example, HCoV.HKU1.C.N5 (GenBank
Accession No. DQ339101), MHV.A59 (GenBank Accession No. NC 001846),
PHEV.VW572 (GenBank Accession No. NC 007732), HCoV.OC43.ATCC.VR.759
(GenBank Accession No. NC_005147), or bovine enteric coronavirus
(BCoV.ENT) (GenBank Accession No. NC_003045).
[0396] Alternatively, the S proteins of CoV to which the antibodies
or antigen-binding antibody fragments of the present disclosure may
specifically bind may include, for example, BtCoV.HKU9.2 (GenBank
Accession No. EF065514), BtCoV.HKU9.1 (GenBank Accession No.
NC_009021), BtCoV.HkU9.3 (GenBank Accession No. EF065515), or
BtCoV.HKU9.4 (GenBank Accession No. EF065516).
[0397] In some instances, an anti-CoV-S antibody or antigen-binding
fragment thereof according to the disclosure binds to CoV-S (e.g.,
SARS-CoV-S and/or SARS-CoV-2-S, and/or any of the CoV S proteins
listed above) with a dissociation constant (KD) of (i) 100 nM or
lower; (ii) about 10 nM or lower; (iii) about 1 nM or lower; (iv)
about 100 pM or lower; (v) about 10 pM or lower; (vi) about 1 pM or
lower; or (vii) about 0.1 pM or lower.
[0398] The present disclosure provides exemplary antibodies or
antigen-binding fragments thereof that bind CoV-S, including human
CoV-S, which optionally may be affinity-matured. Other antibodies
or antigen-binding fragments thereof that bind CoV-S, including
those having different CDRs, and epitopic specificity may be
obtained using the disclosure of the present specification, and
using methods that are generally known in the art. Such antibodies
and antigen-binding fragments thereof antagonize the biological
effects of CoV-S in vivo and therefore are useful in treating or
preventing COV-S-related conditions including, particularly
coronavirus infection. In preferred embodiments, the antibody or
antigen-binding fragment thereof according to the disclosure
comprises one or more CDRs, a V.sub.L chain and/or V.sub.H chain of
the anti-CoV-S antibodies and antigen-binding fragments thereof
described herein.
[0399] In some embodiments, an anti-CoV-S antibody or
antigen-binding fragment thereof according to the disclosure will
interfere with, block, reduce, or modulate the interaction between
COV-S and its receptor(s) (e.g., ACE2, CD209L, L-SIGN, DPP4, or
CD26) on host cells or a S protein-priming protein on host cells
(e.g., TMPRSS2). If binding of the S protein to its receptor is
blocked or reduced, CoV virions may be prohibited from entering the
cells, i.e., infection to further cells is prevented. Also, if the
S protein is prevented from binding to a S protein-priming protein,
the S protein would not be activated and therefore the host cell
entry via the receptor may be reduced, i.e., infection to further
cells is prevented.
[0400] In some instance, an anti-CoV-S antibody or antigen-binding
fragment thereof according to the disclosure is "neutralizing",
e.g., it substantially or totally prevents the specific interaction
of CoV-S with the host receptors or priming protein. As a result,
CoV virions may be substantially or totally cleared by immune cells
of the host, such as phagocytes via, for example, Fc receptor
mediated phagocytosis or mere phagocytosis due to increased time of
virions outside the cells. In some embodiments, the antibody or
antigen-binding fragment thereof neutralizes CoV-S, e.g., by
remaining bound to CoV-S in a location and/or manner that prevents
CoV-S from specifically binding to its receptor or priming protein
on host cells. As a result, CoV virions may be substantially or
totally prevented from entering the cells, i.e. infection to
further cells is prevented. In certain embodiments, an anti-CoV-S
antibody or antigen-binding fragment thereof according to the
disclosure neutralizes CoV (e.g., SARS-CoV and/or SARS-CoV-2) at an
IC50 of about 100 nM or lower, of about 50 nM or lower, of about 20
nM or lower, of about 10 nM or lower, of about 5 nM or lower, of
about 2 nM or lower, of about 1 nM or lower, of about 500 pM or
lower, of about 200 pM or lower, of about 100 pM or lower, of about
50 pM or lower, of about 20 pM or lower, of about 10 pM or lower,
of about 5 pM or lower, of about 2 pM or lower, or of about 1 pM or
lower, or at an IC50 of about 500 ng/mL or lower, of about 200
ng/mL or lower, of about 100 ng/mL or lower, of about 50 ng/mL or
lower, at about 20 ng/mL or lower, at about 10 ng/mL or lower, at
about 20 ng/mL or lower, at about 10 mg/mL or lower, at about 5
ng/mL or lower, at about 2 ng/mL or lower, or at about 1 ng/mL or
lower, in vitro, as measured by any of the neutralization assays
described in Examples herein.
[0401] In some instances, an anti-CoV-S antibody or antigen-binding
fragment thereof according to the disclosure or cocktail thereof,
when administered to a coronavirus infected host or one susceptible
to coronavirus infection such as a health care worker may promote a
neutralization response in the host against the coronavirus which
is sufficient to permit the host to be able to mount an effective
cell-mediated immune response against the virus, e.g., T
cell-mediated or cytokine-mediated immune response against the
coronavirus and/or to be more responsive to other treatment methods
such as drugs, antivirals or other biologics.
[0402] As mentioned, the anti-CoV-S antibodies or antigen-binding
fragments thereof according to the disclosure have a variety of
uses. For example, the subject antibodies and fragments can be
useful in prophylactic or therapeutic applications, as well as
diagnostically in binding assays. The subject anti-CoV-S antibodies
or antigen-binding fragments thereof are useful for affinity
purification of CoV-S, in particular human CoV-S or its ligands and
in screening assays to identify other antagonists of CoV-S
activity. Some of the antibodies or antigen-binding fragments
thereof are useful for inhibiting binding of CoV-S to its
receptor(s) (e.g., ACE2, CD209L, L-SIGN, DPP4, or CD26) on host
cells or a S protein-priming protein on host cells (e.g., TMPRSS2)
or inhibiting COV-S-mediated activities and/or biological
effects.
[0403] As used herein, the term "one or more biological effects
associated with COV-S refers to any biological effect mediated,
induced, or otherwise attributable to COV-S, e.g., binding
properties, functional properties, and other properties of
biological significance. Non-limiting exemplary biological effects
of COV-S include COV-S binding to its receptor(s) (e.g., ACE2,
CD209L, L-SIGN, DPP4, or CD26) on host cells or a S protein-priming
protein on host cells (e.g., TMPRSS2), activation of host cells for
allowing virus entry, activation of immune cells as a result of the
entry of CoV into the cell, e.g., via presentation of CoV
antigen(s) on the host cells' MHC molecule, and resulting
inflammation. The subject anti-CoV-S antibodies are capable of
inhibiting one, a combination of, or all of these exemplary CoV-S
biological activities. For example, the anti-CoV-S antibodies and
antigen-binding fragments thereof provided herein may neutralize
CoV virions or reduce the infectivity of CoV virions.
[0404] The antibody or antigen-binding fragment thereof according
to the disclosure can be used in a variety of therapeutic
applications. For example, in some embodiments the anti-CoV-S
antibody or antigen-binding fragment thereof are useful for
treating conditions associated with CoV-S, such as, but not limited
to, symptoms associated with CoV infection. The CoV may be any CoV,
including SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-HKU1, HCoV-OC43,
HCoV-229E, and HCoV-NL63, and also may be any of the CoV species
listed above herein.
[0405] Specific examples of CoV infection-associated symptoms are
fever, cough, dry cough, shortness of breath or difficulty of
breath, fatigue, aches, runny nose, congestion, sore throat,
conjunctivitis, chest pain, headache, muscle ache, chills, loss of
smell, and loss of taste, and gastrointestinal symptoms including
diarrhea. Complications and/or diseases/disorders associated with
coronavirus infection may include, for example, bronchitis,
pneumonia, respiratory failure, acute respiratory failure, organ
failure, multi-organ system failure, pediatric inflammatory
multisystem syndrome, acute respiratory distress syndrome (a severe
lung condition that causes low oxygen in the blood and organs),
blood clots, cardiac conditions, myocardial injury, myocarditis,
heart failure, cardiac arrest, acute myocardial infarction,
dysrhythmias, venous thromboembolism, post-intensive care syndrome,
shock, anaphylactic shock, cytokine release syndrome, septic shock,
disseminated intravascular coagulation, ischemic stroke,
intracerebral hemorrhage, microangiopathic thrombosis, psychosis,
seizure, nonconvulsive status epilepticus, traumatic brain injury,
stroke, anoxic brain injury, encephalitis, posterior reversible
leukoencephalopathy, necrotizing encephalopathy, post-infectious
encephalitis, autoimmune mediated encephalitis, acute disseminated
encephalomyelitis, acute kidney injury, acute liver injury,
pancreatic injury, immune thrombocytopenia, subacute thyroiditis,
gastrointestinal complications, aspergillosis, increased
susceptibility to infection with another virus or bacteria, and/or
pregnancy-related complications. Certain diseases and conditions,
such as high blood pressure, type 1 diabetes, liver disease,
overweight, chronic lung diseases including cystic fibrosis,
pulmonary fibrosis, and asthma, compromised immune system due to
transplant, use of an immunosuppressant, or HIV infection, and
brain and nervous system condition, may increase the risk of CoV
infection-associated complications and diseases.
[0406] The subject anti-CoV-S antibodies and antigen-binding
fragments thereof may be used alone or in association with other
active agents or drugs, including other biologics, to treat any
subject in which blocking, inhibiting, or neutralizing the in vivo
effect of CoV-S or blocking or inhibiting the interaction of CoV-S
and its receptor(s) (e.g., ACE2, CD209L, L-SIGN, DPP4, or CD26) on
host cells or a S protein-priming protein on host cells (e.g.,
TMPRSS2), is therapeutically desirable. In some embodiment, the
subject anti-CoV-S antibody and antigen-binding fragment thereof,
e.g., ADI-58125, may be used in combination with a second antibody,
or antigen-binding fragment thereof, wherein the second antibody,
or antigen-binding fragment thereof, is selected from the group
consisting of ADI-58122, ADI-58127, ADI-58129, ADI-58131, or a
combination thereof. In some embodiment, the second antibody, or
antigen-binding fragment thereof, is ADI-58122. In one embodiment,
the second antibody, or antigen-binding fragment thereof, is
ADI-58127. In one embodiment, the second antibody, or
antigen-binding fragment thereof, is ADI-58129. In one embodiment,
the second antibody, or antigen-binding fragment thereof, is
ADI-58131.
[0407] Exemplary anti-CoV antibodies and antigen-binding fragments
thereof according to the disclosure, and the specific CDRs thereof
are identified in this section. For convenience, each exemplified
antibody or antigen-binding fragment thereof, and corresponding
sequences are separately identified by a specific nomenclature, as
shown in FIGS. 1, 2 and 36.
[0408] The anti-CoV-S antibodies and antigen-binding fragments
thereof comprising the disclosure have binding affinity for CoV-S,
such as SARS-CoV-S or SARS-CoV-S2. Some antibodies of the present
disclosure bind to SARS-CoV-S or SARS-CoV-S2 with a similar K.sub.D
(M), while some antibodies of the present disclosure bind to
SARS-CoV-S with a lower K.sub.D (M) (i.e., higher affinity) than to
SARS-CoV-S2, and some antibodies of the present disclosure bind to
SARS-CoV-S-2 with a lower K.sub.D (M) (i.e., higher affinity) than
to SARS-CoV-S. Affinities to different CoV-S proteins for
antibodies are provided in FIGS. 7-12 and 14.
C. Anti-CoV-S Antibody Polypeptide Sequences and Nucleic Acid
Sequences Encoding Thereof
Antibodies Disclosed Herein
[0409] Anti-CoV-S antibodies, and antigen-binding fragments
thereof, specifically provided herein include: antibodies
ADI-55688, ADI-55689, ADI-55690, ADI-55691, ADI-55692, ADI-55693,
ADI-55694, ADI-55695, ADI-55696, ADI-55697, ADI-55698, ADI-55699,
ADI-55700, ADI-55701, ADI-55702, ADI-55703, ADI-55704, ADI-55705,
ADI-55706, ADI-55707, ADI-55708, ADI-55709, ADI-55710, ADI-55711,
ADI-55712, ADI-55713, ADI-55714, ADI-55715, ADI-55716, ADI-55717,
ADI-55718, ADI-55719, ADI-55721, ADI-55722, ADI-55723, ADI-55724,
ADI-55725, ADI-55726, ADI-55727, ADI-55728, ADI-55729, ADI-55730,
ADI-55731, ADI-55732, ADI-55733, ADI-55734, ADI-55735, ADI-55736,
ADI-55737, ADI-55738, ADI-55739, ADI-55740, ADI-55741, ADI-55742,
ADI-55743, ADI-55744, ADI-55745, ADI-55746, ADI-55747, ADI-55748,
ADI-55749, ADI-55750, ADI-55751, ADI-55752, ADI-55753, ADI-55754,
ADI-55755, ADI-55756, ADI-55757, ADI-55758, ADI-55720, ADI-55760,
ADI-55761, ADI-55762, ADI-55763, ADI-55765, ADI-55766, ADI-55767,
ADI-55769, ADI-55770, ADI-55771, ADI-55775, ADI-55776, ADI-55777,
ADI-55950, ADI-55951, ADI-55952, ADI-55953, ADI-55954, ADI-55955,
ADI-55956, ADI-55957, ADI-55958, ADI-55959, ADI-55960, ADI-55961,
ADI-55962, ADI-55963, ADI-55964, ADI-55965, ADI-55966, ADI-55967,
ADI-55968, ADI-55969, ADI-55970, ADI-55972, ADI-55973, ADI-55974,
ADI-55975, ADI-55976, ADI-55977, ADI-55978, ADI-55979, ADI-55980,
ADI-55981 ADI-55982, ADI-55984, ADI-55986, ADI-55988, ADI-55989,
ADI-55990, ADI-55992, ADI-55993, ADI-55994, ADI-55995, ADI-55996,
ADI-55997, ADI-55998, ADI-55999, ADI-56000, ADI-56001, ADI-56002,
ADI-56003, ADI-56004, ADI-56005 ADI-56006, ADI-56007, ADI-56008,
ADI-56009, ADI-56010, ADI-56011, ADI-56012, ADI-56013, ADI-56014,
ADI-56015, ADI-56016, ADI-56017, ADI-56018, ADI-56019, ADI-56020,
ADI-56021, ADI-56022, ADI-56023, ADI-56024, ADI-56025, ADI-56026,
ADI-56027, ADI-56028, ADI-56029, ADI-56030, ADI-56031, ADI-56032,
ADI-56033, ADI-56034, ADI-56035, ADI-56037, ADI-56038, ADI-56039,
ADI-56040, ADI-56041, ADI-56042, ADI-56043, ADI-56044, ADI-56045,
ADI-56046, ADI-56047, ADI-56048, ADI-56049, ADI-56050, ADI-56051,
ADI-56052, ADI-56053, ADI-56054, ADI-56055, ADI-56056, ADI-56057,
ADI-56058, ADI-56059, ADI-56061, ADI-56062, ADI-56063, ADI-56064,
ADI-56065, ADI-56066, ADI-56067, ADI-56068, ADI-56069, ADI-56070,
ADI-56071, ADI-56072, ADI-56073, ADI-56074, ADI-56075 ADI-56076,
ADI-56078, ADI-56079, ADI-56080, ADI-56081, ADI-56082, ADI-56083,
ADI-56084, ADI-57983 (with primer mutation), ADI-57978 (with primer
mutation), ADI-56868 (with primer mutation), ADI-56443 (with primer
mutation), ADI-56479 (with primer mutation), ADI-58120, ADI-58121,
ADI-58122, ADI-58123, ADI-58124, ADI-58125, ADI-58126, ADI-58127,
ADI-58128, ADI-58129, ADI-58130, ADI-58131, ADI-58130_LCN30cQ, or
ADI-59988, as shown in FIGS. 1, 2, and 36, and antigen-binding
fragments thereof. Any Fc variant including but not limited to
those specifically disclosed in Table 1 may be used in combination
with any of the variable sequences disclosed herein. In some
embodiments, the Fc variant is an LA variant and comprises the
amino acid sequence of SEQ ID NO: 13. In one embodiment, the
antibody ADI-58125 comprises an Fc variant of SEQ ID NO: 13.
[0410] FIGS. 1, 2 and 36 show the SEQ ID NOs assigned to individual
amino acid sequences of the VH, VH FR1, VH CDR1, VH FR2, VH CDR2,
VH FR3, VH CDR3, VH FR4, VL, VL FR1, VL CDR1, VL FR2, VL CDR2, VL
FR3, VL CDR3, and VL FR4 for individual antibodies, and the SEQ ID
NOs assigned to the nucleic acid sequences of the VH and VL of
individual antibodies.
[0411] For example, for antibody ADI-55688:
[0412] (i) the VH of ADI-55688 comprises the amino acid sequence of
SEQ ID NO: 102;
[0413] (ii) the VH FR1 of ADI-55688 comprises the amino acid
sequence of SEQ ID NO: 103;
[0414] (iii) the VH CDR1 of ADI-55688 comprises the amino acid
sequence of SEQ ID NO: 104;
[0415] (iv) the VH FR2 of ADI-55688 comprises the amino acid
sequence of SEQ ID NO: 105;
[0416] (v) the VH CDR2 of ADI-55688 comprises the amino acid
sequence of SEQ ID NO: 106;
[0417] (vi) the VH FR3 of ADI-55688 comprises the amino acid
sequence of SEQ ID NO: 107;
[0418] (vii) the VH CDR3 of ADI-55688 comprises the amino acid
sequence of SEQ ID NO: 108;
[0419] (viii) the VH FR4 of ADI-55688 comprises the amino acid
sequence of SEQ ID NO: 109;
[0420] (ix) the VH of ADI-55688 is encoded by the nucleic acid
sequence of SEQ ID NO: 110;
[0421] (x) the VL of ADI-55688 comprises the amino acid sequence of
SEQ ID NO: 112;
[0422] (xi) the VL FR1 of ADI-55688 comprises the amino acid
sequence of SEQ ID NO: 113;
[0423] (xii) the VL CDR1 of ADI-55688 comprises the amino acid
sequence of SEQ ID NO: 114;
[0424] (xiii) the VL FR2 of ADI-55688 comprises the amino acid
sequence of SEQ ID NO: 115;
[0425] (xiv) the VL CDR2 of ADI-55688 comprises the amino acid
sequence of SEQ ID NO: 116;
[0426] (xv) the VL FR3 of ADI-55688 comprises the amino acid
sequence of SEQ ID NO: 117;
[0427] (xvi) the VL CDR3 of ADI-55688 comprises the amino acid
sequence of SEQ ID NO: 118.
[0428] (xvii) the VL FR4 of ADI-55688 comprises the amino acid
sequence of SEQ ID NO: 119; and
[0429] (xviii) the VL of ADI-55688 is encoded by the nucleic acid
sequence of SEQ ID NO: 120.
[0430] Analogously, for antibody ADI-55689:
[0431] (i) the VH of ADI-55689 comprises the amino acid sequence of
SEQ ID NO: 202;
[0432] (ii) the VH FR1 of ADI-55689 comprises the amino acid
sequence of SEQ ID NO: 203;
[0433] (iii) the VH CDR1 of ADI-55689 comprises the amino acid
sequence of SEQ ID NO: 204;
[0434] (iv) the VH FR2 of ADI-55689 comprises the amino acid
sequence of SEQ ID NO: 205;
[0435] (v) the VH CDR2 of ADI-55689 comprises the amino acid
sequence of SEQ ID NO: 206;
[0436] (vi) the VH FR3 of ADI-55689 comprises the amino acid
sequence of SEQ ID NO: 207;
[0437] (vii) the VH CDR3 of ADI-55689 comprises the amino acid
sequence of SEQ ID NO: 208;
[0438] (viii) the VH FR4 of ADI-55689 comprises the amino acid
sequence of SEQ ID NO: 209;
[0439] (ix) the VH of ADI-55689 is encoded by the nucleic acid
sequence of SEQ ID NO: 210;
[0440] (x) the VL of ADI-55689 comprises the amino acid sequence of
SEQ ID NO: 212;
[0441] (xi) the VL FR1 of ADI-55689 comprises the amino acid
sequence of SEQ ID NO: 213;
[0442] (xii) the VL CDR1 of ADI-55689 comprises the amino acid
sequence of SEQ ID NO: 214;
[0443] (xiii) the VL FR2 of ADI-55689 comprises the amino acid
sequence of SEQ ID NO: 215;
[0444] (xiv) the VL CDR2 of ADI-55689 comprises the amino acid
sequence of SEQ ID NO: 216;
[0445] (xv) the VL FR3 of ADI-55689 comprises the amino acid
sequence of SEQ ID NO: 217;
[0446] (xvi) the VL CDR3 of ADI-55689 comprises the amino acid
sequence of SEQ ID NO: 218;
[0447] (xvii) the VL FR4 of ADI-55689 comprises the amino acid
sequence of SEQ ID NO: 219; and
[0448] (xviii) the VL of ADI-55689 is encoded by the nucleic acid
sequence of SEQ ID NO: 220.
[0449] The corresponding amino acid and nucleic acid sequences and
sequence identifiers for such sequences and isotype for all other
antibodies are disclosed in FIGS. 1, 2 and 36.
Variations of the Disclosed Antibodies and Polynucleotide Sequences
Encoding Such Variations
[0450] In one embodiment, disclosed herein are anti-CoV-S
antibodies or antigen-binding antibody fragments comprising (i) a
VH CDR that is same as the VH CDR3 of, (ii) a VH CDR3 and VL CDR3,
both of which as same as both of the VH CDR3 and the VL CDR3 of,
(iii) at least 1, 2, 3, 4, 5, or 6 CDRs that are same as the
corresponding CDR(s) of, or (iv) 6 CDRs that are all the same as
the 6 CDRs of any one of the disclosed antibodies described herein
and in FIGS. 1, 2 and 36.
[0451] In further embodiments, disclosed herein are anti-CoV-S
antibodies or antigen-binding antibody fragments which optionally
may be affinity-matured, comprising one of the CDR requirements
(i)-(iv) of the immediately above paragraph, further wherein (a)
the VH comprises an amino acid sequence with at least 80, 85, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to
the amino acid sequence of the VH of, and (b) the VL comprises an
amino acid sequence with at least 80, 85, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, or 100% sequence identity to the amino acid
sequence of the VL of any one of the disclosed antibodies described
herein and in FIGS. 1, 2 and 36.
[0452] In further embodiments, the disclosure contemplates
anti-CoV-S antibodies or antigen-binding antibody fragments which
optionally may be affinity-matured, comprising one of the VH and VL
requirements (i)-(iv) of the immediately above paragraph, further
wherein (a) the heavy chain comprises an amino acid sequence with
at least 80, 85, 90, 91, 92, 93, 94 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of the heavy chain of,
and (b) the light chain comprises an amino acid sequence with at
least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%
sequence identity to the amino acid sequence of the light chain of
any one of the disclosed antibodies as described herein and in
FIGS. 1, 2 and 36.
[0453] In further embodiments, the disclosure contemplates
anti-CoV-S antibodies or antigen-binding antibody fragments which
optionally may be affinity-matured, comprising one of the CDR
requirements (i)-(iv) of the immediately above paragraph, further
wherein (a) the VH is identical to the VH of, and (b) the VL is
identical to the VL of any one of the disclosed antibodies
described herein and in FIGS. 1, 2 and 36.
[0454] In other embodiments, the disclosure includes antibodies and
antigen-binding fragments which optionally may be affinity-matured,
having binding specificity to COV-S that bind the same epitope as
one of antibodies described herein and in FIGS. 1, 2 and 36.
[0455] In other embodiments, the disclosure includes antibodies and
antigen-binding fragments having binding specificity to COV-S,
which optionally may be affinity-matured, that bind the same
epitope as any one of antibodies described herein and in FIGS. 1, 2
and 36.
[0456] In other embodiments, the anti-CoV-S antibodies and
antigen-binding fragments optionally may be affinity-matured,
comprise, or alternatively consist of, combinations of one or more
of the FRs, CDRs, the VH and VL sequences, and the heavy chain and
light chain sequences set forth above, including all of them, or
sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identical thereto.
[0457] In a further embodiment, antigen-binding fragments comprise,
or alternatively consist of, Fab fragments having binding
specificity for COV-S. The Fab fragment preferably includes the VH
and the VL sequence of any one of antibodies as described herein
and in FIGS. 1, 2 and 36, or sequences that are at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. This
embodiment further includes Fabs containing additions, deletions,
and variants of such VH and VL sequence while retaining binding
specificity for COV-S.
[0458] In some embodiments, Fab fragments may be produced by
enzymatic digestion (e.g., papain) of the parent full antibody. In
another embodiment, anti-CoV-S antibodies, such as anyone of
antibodies as described herein and in FIGS. 1, 2 and 36, and Fab
fragments thereof may be produced via expression in mammalian
cells, such as CHO, NSO, or HEK 293 cells, fungal, insect, or
microbial systems, such as yeast cells.
[0459] In additional embodiments, disclosed herein are
polynucleotides encoding antibody polypeptides having binding
specificity to COV-S, including the VH and VL of any one of
antibodies as described herein and in FIGS. 1, 2 and 36, as well as
fragments, variants, optionally affinity-matured variants, and
combinations of one or more of the FRs, CDRs, the VH and VL
sequences, and the heavy chain and light chain sequences set forth
above, including all of them, or sequences that are at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
thereto.
[0460] In other embodiments, the disclosure contemplates isolated
anti-CoV-S antibodies and antigen binding fragments comprising (i)
a VH which is same as the VH of any one of antibodies as described
herein and in FIGS. 1, 2 and 36; and (ii) a VL which is same as the
VL of another antibody as described herein and in FIGS. 1, 2 and
36, or a variant thereof, wherein optionally one or more of the
framework region residues ("FR residues") and/or CDR residues in
said VH or V.sub.L polypeptide has been substituted with another
amino acid residue resulting in an anti-CoV-S antibody that
specifically binds COV-S.
[0461] The disclosure also includes humanized, primatized and other
chimeric forms of these antibodies. The chimeric and humanized
antibodies may include an Fc derived from IgG1, IgG2, IgG3, or IgG4
constant regions.
[0462] In some embodiments, the chimeric or humanized antibodies or
fragments or VH or VL polypeptides originate or are derived from
one or more human antibodies, e.g., a human antibody identified
from a clonal human B cell population.
[0463] In some aspects, the disclosure provides vectors comprising
a nucleic acid molecule encoding an anti-CoV-S antibody or fragment
thereof as disclosed herein. In some embodiments, the disclosure
provides host cells comprising a nucleic acid molecule encoding an
anti-CoV-S antibody or fragment thereof as disclosed herein.
[0464] In some aspects, the disclosure provides isolated antibodies
or antigen binding fragments thereof that competes for binding to
CoV-S with an antibody or antigen binding fragment thereof
disclosed herein.
[0465] In some aspects, the disclosure provides a nucleic acid
molecule encoding any of the antibodies or antigen binding
fragments disclosed herein.
[0466] In some aspects, the disclosure provides a pharmaceutical or
diagnostic composition comprising at least one antibody or antigen
binding fragment thereof as disclosed herein.
[0467] In some aspects, the disclosure provides a method for
treating or preventing a condition associated with elevated CoV-S
levels in a subject, comprising administering to a subject in need
thereof an effective amount of at least one isolated antibody or
antigen binding fragment thereof as disclosed herein.
[0468] In some aspects, the disclosure provides a method of
inhibiting binding of COV-S to its receptor (e.g., ACE2, L-SIGN,
CD209L, DPP4, CD26) or an S protein-priming protein (e.g., TMPRSS2)
in a subject comprising administering an effective amount of at
least one antibody or antigen binding fragment thereof as disclosed
herein. For example, administering one or more of ADI-55689;
ADI-55993; ADI-56000; ADI-56046; ADI-56010; ADI-55688; ADI-56032;
ADI-55690; and ADI-55951 may inhibit binding of COV-S to its
receptor, e.g., ACE2.
[0469] In some aspects, the disclosure provides an antibody or
antigen binding fragment thereof that selectively binds to CoV-S,
wherein the antibody or antigen binding fragment thereof binds to
CoV-S with a K.sub.D of less than or equal to 5.times.10.sup.-5 M,
10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M, 5.times.10.sup.-7 M,
10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9 M,
10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10 M, 5.times.10.sup.-11
M, 10.sup.-11 M, 5.times.10.sup.-12 M, 10.sup.-12 M,
5.times.10.sup.-13 M, or 10.sup.-13 M; preferably, with a K.sub.D
of less than or equal to 5.times.10.sup.-10 M, 10.sup.-10 M,
5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M, or
10.sup.-12 M; more preferably, with a K.sub.D that is less than
about 100 pM, less than about 50 pM, less than about 40 pM, less
than about 25 pM, less than about 1 pM, between about 10 pM and
about 100 pM, between about 1 pM and about 100 pM, or between about
1 pM and about 10 pM. Preferably, the anti-CoV-S antibody or
antigen binding fragment has cross-reactivity to the S protein of
CoV other than SARS-CoV-S or SARS-CoV-2-S.
[0470] The inventive antibodies and antigen binding fragments
thereof may be modified post-translationally to add effector
moieties such as chemical linkers, detectable moieties such as for
example fluorescent dyes, enzymes, substrates, bioluminescent
materials, radioactive materials, and chemiluminescent moieties, or
functional moieties such as for example streptavidin, avidin,
biotin, a cytotoxin, a cytotoxic agent, and radioactive
materials.
[0471] Antibodies and antigen binding fragments thereof may also be
chemically modified to provide additional advantages such as
increased solubility, stability and circulating time (in vivo
half-life) of the polypeptide, or decreased immunogenicity (See
U.S. Pat. No. 4,179,337). The chemical moieties for derivatization
may be selected from water soluble polymers such as polyethylene
glycol, ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol, and the like.
The antibodies and fragments thereof may be modified at random
positions within the molecule, or at predetermined positions within
the molecule and may include one, two, three, or more attached
chemical moieties.
[0472] The polymer may be of any molecular weight, and may be
branched or unbranched. For polyethylene glycol, the preferred
molecular weight is between about 1 kDa and about 100 kDa (the term
"about" indicating that in preparations of polyethylene glycol,
some molecules will weigh more, some less, than the stated
molecular weight) for ease in handling and manufacturing. Other
sizes may be used, depending on the desired therapeutic profile
(e.g., the duration of sustained release desired, the effects, if
any on biological activity, the ease in handling, the degree or
lack of antigenicity and other known effects of the polyethylene
glycol to a therapeutic protein or analog). For example, the
polyethylene glycol may have an average molecular weight of about
200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,
5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000,
10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000,
14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000,
18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000,
50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000,
90,000, 95,000, or 100,000 kDa. Branched polyethylene glycols are
described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et
al., Appl. Biochem. Biotechnol., 56:59-72 (1996); Vorobjev et al.,
Nucleosides and Nucleotides, 18:2745-2750 (1999); and Caliceti et
al., Bioconjug. Chem., 10:638-646 (1999), the disclosures of each
of which are incorporated herein by reference.
[0473] There are a number of attachment methods available to those
skilled in the art (See e.g., EP 0 401 384, herein incorporated by
reference, disclosing a method of coupling PEG to G-CSF; and Malik
et al., Exp. Hematol., 20:1028-1035 (1992) (reporting pegylation of
GM-CSF using tresyl chloride)). For example, polyethylene glycol
may be covalently bound through amino acid residues via a reactive
group, such as, a free amino or carboxyl group. Reactive groups are
those to which an activated polyethylene glycol molecule may be
bound. The amino acid residues having a free amino group may
include lysine residues and the N-terminal amino acid residues;
those having a free carboxyl group may include aspartic acid
residues glutamic acid residues and the C-terminal amino acid
residue. Sulfhydryl groups may also be used as a reactive group for
attaching the polyethylene glycol molecules. Preferred for
therapeutic purposes is attachment at an amino group, such as
attachment at the N-terminus or lysine group.
[0474] As described above, polyethylene glycol may be attached to
proteins via linkage to any of a number of amino acid residues. For
example, polyethylene glycol can be linked to polypeptides via
covalent bonds to lysine, histidine, aspartic acid, glutamic acid,
or cysteine residues. One or more reaction chemistries may be
employed to attach polyethylene glycol to specific amino acid
residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or
cysteine) or to more than one type of amino acid residue (e.g.,
lysine, histidine, aspartic acid, glutamic acid, cysteine and
combinations thereof).
[0475] Alternatively, antibodies or antigen binding fragments
thereof having increased in vivo half-lives may be produced via
fusion with albumin (including but not limited to recombinant human
serum albumin or fragments or variants thereof (See, e.g., U.S.
Pat. No. 5,876,969, EP 0 413 622, and U.S. Pat. No. 5,766,883,
herein incorporated by reference in their entirety)), or other
circulating blood proteins such as transferrin or ferritin. In a
preferred embodiment, polypeptides and/or antibodies of the present
disclosure (including fragments or variants thereof) are fused with
the mature form of human serum albumin (i.e., amino acids 1-585 of
human serum albumin as shown in FIGS. 1 and 2 of EP 0 322 094)
which is herein incorporated by reference in its entirety.
Polynucleotides encoding fusion proteins of the disclosure are also
encompassed by the disclosure.
[0476] Regarding detectable moieties, further exemplary enzymes
include, but are not limited to, horseradish peroxidase,
acetylcholinesterase, alkaline phosphatase, beta-galactosidase, and
luciferase. Further exemplary fluorescent materials include, but
are not limited to, rhodamine, fluorescein, fluorescein
isothiocyanate, umbelliferone, dichlorotriazinylamine,
phycoerythrin, and dansyl chloride. Further exemplary
chemiluminescent moieties include, but are not limited to, luminol.
Further exemplary bioluminescent materials include, but are not
limited to, luciferin and aequorin. Further exemplary radioactive
materials include, but are not limited to, Iodine 125 (.sup.125I),
Carbon 14 (.sup.14C), Sulfur 35 (.sup.35S), Tritium (.sup.3H) and
Phosphorus 32 (.sup.32P).
[0477] Methods are known in the art for conjugating an antibody or
antigen binding fragment thereof to a detectable moiety and the
like, such as for example those methods described by Hunter et al.,
Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974);
Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J.,
Histochem. and Cytochem., 30:407 (1982).
[0478] Embodiments described herein further include variants and
equivalents that are substantially homologous to the antibodies,
antibody fragments, diabodies, SMIPs, camelbodies, nanobodies,
IgNAR, polypeptides, variable regions, and CDRs set forth herein.
These may contain, e.g., conservative substitution mutations,
(i.e., the substitution of one or more amino acids by similar amino
acids). For example, conservative substitution refers to the
substitution of an amino acid with another within the same general
class, e.g., one acidic amino acid with another acidic amino acid,
one basic amino acid with another basic amino acid, or one neutral
amino acid by another neutral amino acid. The intent of a
conservative amino acid substitution is well known in the art.
[0479] In other embodiments, the disclosure contemplates
polypeptide sequences having at least 90% or greater sequence
homology to any one or more of the polypeptide sequences of antigen
binding fragments, variable regions and CDRs set forth herein. More
preferably, the disclosure contemplates polypeptide sequences
having at least 95% or greater sequence homology, even more
preferably at least 98% or greater sequence homology, and still
more preferably at least 99% or greater sequence homology to any
one or more of the polypeptide sequences of antigen binding
fragments, variable regions, and CDRs set forth herein.
[0480] Methods for determining homology between nucleic acid and
amino acid sequences are well known to those of ordinary skill in
the art.
[0481] In other embodiments, the disclosure further contemplates
the above-recited polypeptide homologs of the antigen binding
fragments, variable regions and CDRs set forth herein further
having anti-CoV-S activity. Non-limiting examples of anti-CoV-S
activity are set forth herein, e.g., ability to inhibit CoV-S
binding to its receptor such as ACE2 or L-SIGN or an S
protein-priming protein, thereby resulting in the reduced entry of
CoV into cells.
[0482] In other embodiments, the disclosure further contemplates
the generation and use of antibodies that bind any of the foregoing
sequences, including, but not limited to, anti-idiotypic
antibodies. In an exemplary embodiment, such an anti-idiotypic
antibody could be administered to a subject who has received an
anti-CoV-S antibody to modulate, reduce, or neutralize, the effect
of the anti-CoV-S antibody. Such antibodies could also be useful
for treatment of an autoimmune disease characterized by the
presence of anti-CoV-S antibodies. A further exemplary use of such
antibodies, e.g., anti-idiotypic antibodies, is for detection of
the anti-CoV-S antibodies of the present disclosure, for example to
monitor the levels of the anti-CoV-S antibodies present in a
subject's blood or other bodily fluids. For example, in one
embodiment, the disclosure provides a method of using the
anti-idiotypic antibody to monitor the in vivo levels of said
anti-CoV-S antibody or antigen binding fragment thereof in a
subject or to neutralize said anti-CoV-S antibody in a subject
being administered said anti-CoV-S antibody or antigen binding
fragment thereof.
[0483] The present disclosure also contemplates anti-CoV-S
antibodies comprising any of the polypeptide or polynucleotide
sequences described herein substituted for any of the other
polynucleotide sequences described herein. For example, without
limitation thereto, the present disclosure contemplates antibodies
comprising the combination of any of the VL and VH sequences
described herein, and further contemplates antibodies resulting
from substitution of any of the CDR sequences described herein for
any of the other CDR sequences described herein.
[0484] Another embodiment of the disclosure contemplates these
polynucleotides incorporated into an expression vector for
expression in mammalian cells such as CHO, NSO, or HEK-293 cells,
or in fungal, insect, or microbial systems such as yeast cells. In
one embodiment of the disclosure described herein, Fab fragments
can be produced by enzymatic digestion (e.g., papain) of any one of
antibodies as described herein and in FIGS. 1, 2 and 36, following
expression of the full-length polynucleotides in a suitable host.
In another embodiment, anti-CoV-S antibodies, such as anyone of
antibodies as described herein and in FIGS. 1, 2 and 36, or Fab
fragments thereof, can be produced via expression of the
polynucleotides encoding any one of antibodies as described herein
and in FIGS. 1, 2 and 36, preferably, in certain embodiments,
ADI-57983 (with primer mutation), ADI-57978 (with primer mutation),
ADI-56868 (with primer mutation), ADI-56443 (with primer mutation),
ADI-56479 (with primer mutation), ADI-58120, ADI-58121, ADI-58122,
ADI-58123, ADI-58124, ADI-58125, ADI-58126, ADI-58127, ADI-58128,
ADI-58129, ADI-58130, ADI-58131, ADI-58130_LCN30cQ, or ADI-59988,
in mammalian cells such as CHO, NSO, or HEK 293 cells, fungal,
insect, or microbial systems such as yeast cells.
[0485] Host cells and vectors comprising said polynucleotides are
also contemplated.
[0486] The disclosure further contemplates vectors comprising the
polynucleotide sequences encoding the variable heavy and light
chain polypeptide sequences, as well as the individual CDRs
(hypervariable regions), as set forth herein, as well as host cells
comprising said vector sequences. In one embodiment, the host cells
are mammalian cells, such as CHO cells. In one embodiment, the host
cells are yeast cells.
D. Antibody-Drug Conjugate Comprising Anti-CoV-S Antibody
[0487] In some aspects, the disclosure is further directed to
antibody-drug conjugates (ADCs) comprising (a) any antibody or
antigen-binding antibody fragment described herein; and (b) a drug
conjugated to the antibody or antigen-binding antibody fragment,
either directly or indirectly (e.g., via a linker).
[0488] In some aspects, the drug may be, but not limited to, a
cytotoxic drug, an apoptotic drug, an immunostimulatory drug, an
anti-microbial drug, an antibacterial drug or vaccine, an antiviral
drug, antihelminth drug, antiparasitic drug, an anti-inflammatory
drug, antihistamine, an anti-fibrotic drug, an immunosuppressive
drug, a steroid, a bronchodilator, a beta blocker, an ACE
inhibitor, an enzyme, a serine protease inhibitor, a toxin, a
radioisotope, a compound, a small molecule, a small molecule
inhibitor, a protein, a peptide, a vector, a plasmid, a viral
particle, a nanoparticle, a DNA molecule, an RNA molecule, an
siRNA, an shRNA, a micro RNA, an oligonucleotide, and an imaging
drug.
[0489] An antiviral drug may be remdesivir, favipiravir, darunavir,
nelfinavir, saquinavir, lopinavir or ritonavir; an antihelminth
drug may be ivermectin; an antiparasite drug may be
hydroxychloroquine, chloroquine, or atovaquone; antibacterial drug
or vaccine may be the tuberculosis vaccine BCG; an
anti-inflammatory drug, may be ciclesonide, a TNF inhibitor (e.g.,
adalimumab), a TNF receptor inhibitor (e.g., etanercept), an IL-6
inhibitor (e.g., clazakizumab), an IL-6 receptor inhibitor (e.g.,
toclizumab), or metamizole; an antihistamine drug may be
bepotastine; an ACE inhibitor may be moexipril; and a drug that
inhibits priming of CoV-S may be a serine protease inhibitor such
as nafamostat.
[0490] The toxin may be a bacterial, fungal, plant, or animal
toxin, or a fragment thereof. Examples include, but are not limited
to, diphtheria A chain, diphtheria toxin, exotoxin A chain, ricin A
chain, abrin A chain, modeccin A chain, alpha sarcin, Aleurites
fordii protein, a dianthin protein, or a Phytolacca Americana
protein.
[0491] The cytotoxic drug or anti-proliferative drug may be, for
example, but is not limited to, doxorubicin, daunorubicin,
cucurbitacin, chaetocin, chaetoglobosin, chlamydocin,
calicheamicin, nemorubicin, cryptophyscin, mensacarcin,
ansamitocin, mitomycin C, geldanamycin, mechercharmycin,
rebeccamycin, safracin, okilactomycin, oligomycin, actinomycin,
sandramycin, hypothemycin, polyketomycin, hydroxyellipticine,
thiocolchicine, methotrexate, triptolide, taltobulin, lactacystin,
dolastatin, auristatin, monomethyl auristatin E (MMAE), monomethyl
auristatin F (MMAF), telomestatin, tubastatin A, combretastatin,
maytansinoid, MMAD, MMAF, DM1, DM4, DTT, 16-GMB-APA-GA, 17-DMAP-GA,
JW 55, pyrrolobenzodiazepine, SN-38, Ro 5-3335, puwainaphycin,
duocarmycin, bafilomycin, taxoid, tubulysin, ferulenol, lusiol A,
fumagillin, hygrolidin, glucopiericidin, amanitin, ansatrienin,
cinerubin, phallacidin, phalloidin, phytosphongosine, piericidin,
poronetin, phodophyllotoxin, gramicidin A, sanguinarine,
sinefungin, herboxidiene, microcolin B, microcystin, muscotoxin A,
tolytoxin, tripolin A, myoseverin, mytoxin B, nocuolin A,
psuedolaric acid B, pseurotin A, cyclopamine, curvulin, colchicine,
aphidicolin, englerin, cordycepin, apoptolidin, epothilone A,
limaquinone, isatropolone, isofistularin, quinaldopeptin,
ixabepilone, aeroplysinin, arruginosin, agrochelin, epothilone, or
a derivative thereof (for example, see Polakis P. et al., Pharmacol
Rev. 2016 January; 68(1):3-19. doi: 10.1124/pr.114.009373) (the
drugs may be obtained from many vendors, including Creative
Biolabs.RTM.).
[0492] The radioisotope may be for example, but is not limited to,
At.sup.211, I.sup.131, In.sup.131, I.sup.125, Y.sup.90, Re.sup.186,
Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32, Pb.sup.212 and
radioactive isotopes of Lu.
[0493] In certain embodiments, the drug may be, but is not limited
to, MMAE or MMAF.
[0494] In some embodiments, the Ab or antigen-binding Ab fragment
is directly conjugated to the drug to form an ADC.
[0495] In some embodiments, the antibody or antigen-binding
antibody fragment is indirectly conjugated to the drug to form an
ADC.
[0496] Any appropriate conjugation method may be used to generate
an ADC (for example, Nolting B. Methods Mol Biol. 2013;
1045:71-100; Jain N. et al., Pharm Res. 2015 November;
32(11):3526-40; Tsuchikama K. et al., Protein Cell. 2018 January;
9(1):33-46; Polakis P. et al., Pharmacol Rev. 2016 January;
68(1):3-19). Examples of methods that may be used to perform
conjugation include, but are not limited to, chemical conjugation
and enzymatic conjugation.
[0497] Chemical conjugation may utilize, for example, but is not
limited to, lysine amide coupling, cysteine coupling, and/or
non-natural amino acid incorporation by genetic engineering.
Enzymatic conjugation may utilize, for example, but is not limited
to, transpeptidation using sortase, transpeptidation using
microbial transglutaminase, and/or N-Glycan engineering.
[0498] In certain aspects, one or more of cleavable linkers may be
used for conjugation. The cleavable linker may enable cleavage of
the drug upon responding to, for example, but not limited to, an
environmental difference between the extracellular and
intracellular environments (pH, redox potential, etc.) or by
specific lysosomal enzymes.
[0499] Examples of the cleavable linker include, but are not
limited to, hydrazone linkers, peptide linkers including cathepsin
B-responsive linkers, such as valine-citrulline (vc) linker,
disulfide linkers such as N-succinimidyl-4-(2-pyridyldithio) (SPP)
linker or N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB)
linker, and pyrophosphate diester linkers.
[0500] Alternatively or simultaneously, one or more of
non-cleavable linkers may be used. Examples of non-cleavable
linkers include thioether linkers, such as N-succinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), and
maleimidocaproyl (me) linkers. Generally, non-cleavable linkers are
more resistant to proteolytic degradation and more stable compared
to cleavable linkers.
E. Chimeric Antigen Receptor Comprising Anti-CoV-S Antigen-Binding
Antibody Fragment
[0501] In some embodiments, a compound specific to CoV-S according
to the present disclosure may be a chimeric antigen receptor (CAR).
In particular, the CARs of the present disclosure comprise an
antigen binding (AB) domain that binds to CoV-S, a transmembrane
(TM) domain, and an intracellular signaling (ICS) domain. In some
embodiments, a CAR may comprise a hinge that joins the AB domain
and said TM domain. In some embodiments, the CAR may comprise one
or more costimulatory (CS) domains.
AB Domain
[0502] A CAR according to the disclosure will comprise an
antigen-binding (AB) domain which binds to COV-S. In some
embodiments, the AB domain of the CAR may comprise any of the
anti-COV-S antigen-binding antibody fragments disclosed herein.
[0503] In some embodiments, the AB domain of the CAR may comprise
any of the antigen-binding domain of any of the anti-COV-S
antibodies disclosed herein.
[0504] In some embodiments, the AB domain of the CAR may comprise
any of the anti-COV-S antibodies, anti-COV-S antigen-binding
antibody fragments, anti-COV-S multi-specific Abs, anti-COV-S
multi-specific antigen-binding antibody fragments, and anti-COV-S
ADCs disclosed herein, or the ABD thereof.
[0505] In some embodiments, the AB domain of the CAR may comprise
an anti-COV-S scFv.
[0506] In some embodiments, the AB domain may comprise an amino
acid sequence at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%
identical to an scFv comprising the VH and VL of any one of
antibodies as described herein and in FIGS. 1, 2 and 36.
[0507] In some aspects, the AB domain may compete for binding to
COV-S with any one of antibodies as described herein and in FIGS.
1, 2 and 36.
Hinge
[0508] In some embodiments, the CAR may comprise a hinge sequence
between the AB domain and the TM domain. One of the ordinary skill
in the art will appreciate that a hinge sequence is a short
sequence of amino acids that facilitates flexibility (see, e.g.
Woof J. M. et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)). The
hinge sequence can be any suitable sequence derived or obtained
from any suitable molecule.
[0509] In some embodiments, the length of the hinge sequence may be
optimized based on the desired length of the extracellular portion
of the CAR, which may be based on the location of the epitope
within the target molecule. For example, if the epitope is in the
membrane proximal region within the target molecule, longer hinges
may be optimal.
[0510] In some embodiments, the hinge may be derived from or
include at least a portion of an immunoglobulin Fc region, for
example, an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region,
an IgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc
region. In certain embodiments, the hinge includes at least a
portion of an IgG1, an IgG2, an IgG3, an IgG4, an IgE, an IgM, or
an IgA immunoglobulin Fc region that falls within its CH2 and CH3
domains. In some embodiments, the hinge may also include at least a
portion of a corresponding immunoglobulin hinge region. In some
embodiments, the hinge is derived from or includes at least a
portion of a modified immunoglobulin Fc region, for example, a
modified IgG1 Fc region, a modified IgG2 Fc region, a modified IgG3
Fc region, a modified IgG4 Fc region, a modified IgE Fc region, a
modified IgM Fc region, or a modified IgA Fc region. The modified
immunoglobulin Fc region may have one or more mutations (e.g.,
point mutations, insertions, deletions, duplications) resulting in
one or more amino acid substitutions, modifications, or deletions
that cause impaired binding of the hinge to an Fc receptor (FcR).
In some aspects, the modified immunoglobulin Fc region may be
designed with one or more mutations which result in one or more
amino acid substitutions, modifications, or deletions that cause
impaired binding of the hinge to one or more FcR including, but not
limited to, Fc.gamma.RI, Fc.gamma.R2A, Fc.gamma.R2B1, Fc.gamma.2B2,
Fc.gamma.3A, Fc.gamma. 3B, Fc.epsilon.RI, Fc.epsilon.R2,
Fc.alpha.RI, Fc.alpha./.mu.R, or FcRn.
[0511] In some aspects, a portion of the immunoglobulin constant
region may serve as a hinge between the AB domain, for example scFv
or nanobody, and the TM domain. The hinge can be of a length that
provides for increased responsiveness of the CAR-expressing cell
following antigen binding, as compared to in the absence of the
hinge. In some examples, the hinge is at or about 12 amino acids in
length or is no more than 12 amino acids in length. Exemplary
hinges include those having at least about 10 to 229 amino acids,
about 10 to 200 amino acids, about 10 to 175 amino acids, about 10
to 150 amino acids, about 10 to 125 amino acids, about 10 to 100
amino acids, about 10 to 75 amino acids, about 10 to 50 amino
acids, about 10 to 40 amino acids, about 10 to 30 amino acids,
about 10 to 20 amino acids, or about 10 to 15 amino acids, and
including any integer between the endpoints of any of the listed
ranges. In some embodiments, a hinge has about 12 amino acids or
less, about 119 amino acids or less, or about 229 amino acids or
less. Exemplary hinges include a CD28 hinge, IgG4 hinge alone, IgG4
hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the
CH3 domain. Exemplary hinges include, but are not limited to, those
described in Hudecek M. et al. (2013) Clin. Cancer Res., 19:3153,
international patent application publication number WO2014031687,
U.S. Pat. No. 8,822,647 or published App. No. US2014/0271635.
[0512] Known hinge sequences include those derived from CD8 a
molecule or a CD28 molecule.
Transmembrane (TM) Domain
[0513] With respect to the TM domain, the CAR can be designed to
comprise a TM domain that is fused to the AB domain of the CAR. A
hinge sequence may be inserted between the AB domain and the TM
domain. TM domains may be derived from a natural or from synthetic
sources. Where the source is natural, the domain may be derived
from any membrane-bound or transmembrane protein. Typically, a TM
domain denotes a single transmembrane a helix of a transmembrane
protein, also known as an integral protein. TM domains e.g., may be
derived from (i.e. comprise at least the transmembrane region(s)
of) CD28, CD3 .epsilon., CD4, CD5, CD8, CD9, CD16, CD22, CD33,
CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, TCR .alpha., TCR
.beta., or CD3 zeta and/or TM domains containing functional
variants thereof such as those retaining a substantial portion of
the structural, e.g., transmembrane, properties thereof.
[0514] Alternatively, the TM domain may be synthetic, in which case
the TM domain will comprise predominantly hydrophobic residues such
as leucine and valine. Preferably a triplet of phenylalanine,
tryptophan and valine will be found at each end of a synthetic TM
domain. A TM domain is generally thermodynamically stable in a
membrane. It may be a single .alpha. helix, a transmembrane .beta.
barrel, a .beta.-helix of gramicidin A, or any other structure.
Transmembrane helices are usually about 20 amino acids in
length.
[0515] A well-used TM domain comprises the TM region of CD28, e.g.,
human CD28. Often, a short oligo- or polypeptide spacer, e.g.,
between 2 and 10 amino acids in length is used to form the linkage
between the TM domain and the ICS domain(s) of the CAR.
Intracellular Signaling (ICS) Domain and Costimulatory (CS)
Domain
[0516] The ICS domain or the cytoplasmic domain of a CAR generally
triggers or elicits activation of at least one of the normal
effector functions of the cell in which the CAR has been placed.
The term "effector function" refers to a specialized function of a
cell. Effector function of a T cell, for example, may be cytolytic
activity or helper activity including the secretion of cytokines.
Thus, the term "intracellular signaling domain" or "ICS domain"
refers to the portion of a protein which transduces the effector
function signal and directs the cell to perform a specialized
function. While usually the entire ICS domain can be employed, in
many cases it is not necessary to use the entire chain. To the
extent that a truncated portion of the intracellular signaling
domain is used, such truncated portion may be used in place of the
intact chain as long as it transduces the effector function signal.
The term "intracellular signaling domain" or "ICS domain" is thus
meant to include any truncated portion of the ICS domain sufficient
to transduce the effector function signal.
[0517] Examples of known ICS domains include the cytoplasmic
sequences of the T cell receptor (TCR) and co-receptors that act in
concert to initiate signal transduction following antigen receptor
engagement, as well as any derivative or variant of these sequences
and any synthetic sequence that has the same functional
capability.
[0518] Signals generated through one ICS domain alone may be
insufficient for full activation of a cell, and a secondary or
costimulatory signal may also be required. In such cases, a
costimulatory domain (CS domain) may be included in the cytoplasmic
portion of a CAR. A CS domain is a domain that transduces such a
secondary or costimulatory signal. In some instances, a CAR of the
present disclosure may comprise two or more CS domains. The CS
domain(s) may be placed upstream of the ICS domain or downstream of
the ICS domain.
[0519] T cell activation can be mediated by two distinct classes of
cytoplasmic signaling sequence: those that initiate
antigen-dependent primary activation through the TCR (primary
cytoplasmic signaling sequences) and those that act in an
antigen-independent manner to provide a secondary or costimulatory
signal (secondary cytoplasmic signaling sequences). Primary
cytoplasmic signaling sequences regulate primary activation of the
TCR complex either in a stimulatory way, or in an inhibitory way.
Primary cytoplasmic signaling sequences that act in a stimulatory
manner may contain signaling motifs which are known as
immunoreceptor tyrosine-based activation motifs or ITAMs. Such a
cytoplasmic signaling sequence may be contained in the ICS or the
CS domain of a CAR.
[0520] Examples of ITAM-containing primary cytoplasmic signaling
sequences include those derived from an ICS domain of a lymphocyte
receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit,
an IL-2 receptor subunit, CD3.zeta., FcR .gamma., FcR.beta.,
CD3.gamma., CD3.delta., CD3.epsilon., CD5, CD22, CD66d, CD79a,
CD79b, CD278 (ICOS), Fc.epsilon. RI, DAP10, and DAP12. A well-used
ICS domain comprises a cytoplasmic signaling sequence derived from
CD3 zeta. In some instances, the CD3(ICS domain may be combined
with one or more of other cytoplasmic domain(s). For example, the
cytoplasmic domain of the CAR can comprise a CD3 .zeta. ICS domain
and a CS domain wherein a CS region refers to a portion of the CAR
comprising the intracellular domain of a costimulatory molecule. A
costimulatory molecule is a cell surface molecule other than an
antigen receptor or their ligands that is required for an efficient
response of lymphocytes to an antigen.
[0521] Examples of co-stimulatory molecules include an MHC class I
molecule, TNF receptor proteins, Immunoglobulin-like proteins,
cytokine receptors, integrins, signaling lymphocytic activation
molecules (SLAM proteins), activating NK cell receptors, a Toll
ligand receptor, B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CD2, CD4, CD5,
CD7, CD8 .alpha., CD8 .beta., CD11a, LFA-1 (CD11a/CD18), CD11b,
CD11c, CD11d, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40,
CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D),
CD103, CRTAM, OX40 (CD134), 4-1BB (CD137), SLAM (SLAMF1, CD150,
IPO-3), CD160 (BY55), SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229),
SLAMF4 (CD244, 2B4), ICOS (CD278), CEACAM1, CDS, CRTAM, DAP10,
GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, IL2R .beta., IL2R .gamma.,
IL7R .alpha., ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX,
ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, NKG2C, NKG2D,
NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG/Cbp, PD-1, PSGL1, SLAMF6
(NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE/RANKL, VLA1, VLA-6, a
ligand that specifically binds with CD83, and the like. The ICS
domain and the CS domain(s) of the CAR may be linked to each other
in a random or specified order, optionally via a short oligo- or
polypeptide linker, e.g., between 2 and 10 amino acids in
length.
Exemplary CAR Constructs
[0522] A CAR construct may comprise the following format: "AB
domain-hinge-TM domain-CS domain-ICS domain."
[0523] CARs may comprise an amino acid sequence at least 80%, at
least 85%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% identical to any of the exemplary
constructs below. In the exemplary constructs below, the
"anti-CoV-S scFv" may be an scFv generated by linking the VH and VL
(in the order of VH-linker-VL or VL-linker-VH) of any one of
anti-CoV-S antibodies as described herein and in FIGS. 1, 2 and
36.
[0524] In some embodiments, a leader sequence (LS) may be placed
upstream of the polynucleotide sequences encoding the CAR. The
leader sequence facilitates the expression of the CAR on the cell
surface.
Further Modification
[0525] CARs according to the present disclosure, nucleotide
sequences encoding the same, vectors encoding the same, and cells
comprising nucleotide sequences encoding said CARs may be further
modified, engineered, optimized, or appended in order to provide or
select for various features. These features may include, but are
not limited to, efficacy, persistence, target specificity, reduced
immunogenicity, multi-targeting, enhanced immune response,
expansion, growth, reduced off-target effect, reduced subject
toxicity, improved target cytotoxicity, improved attraction of
disease alleviating immune cells, detection, selection, targeting,
and the like. For example, the cells may be engineered to express
another CAR, or to have a suicide mechanism, and may be modified to
remove or modify expression of an endogenous receptor or molecule
such as a TCR and/or MHC molecule.
[0526] In some embodiments, the vector or nucleic acid sequence
encoding the CAR further encodes other genes. The vector or nucleic
acid sequence may be constructed to allow for the co-expression of
multiple genes using a multitude of techniques including
co-transfection of two or more plasmids, the use of multiple or
bidirectional promoters, or the creation of bicistronic or
multicistronic vectors. The construction of multicistronic vectors
may include the encoding of IRES elements or 2A peptides, such as
T2A, P2A, E2A, or F2A (for example, see Kim, J. H., et al., "High
cleavage efficiency of a 2A peptide derived from porcine
teschovirus-1 in human cell lines, zebrafish and mice", PLoS One.
2011; 6(4)). The CAR expressing cell may further comprise a
disruption to one or more endogenous genes.
Efficacy
[0527] The CARs of the present disclosure and cells expressing
these CARs may be further modified to improve efficacy against
cells expressing the target molecule. The cells may be cells
expressing COV-S. The cells expressing COV-S may be cancer cells,
vascular cells, or any other target disease-associated cells. In
some embodiments, the improved efficacy may be measured by
increased cytotoxicity against cells expressing the target
molecule, for example cytotoxicity against cancer cells. In some
embodiments, the improved efficacy may also be measured by
increased production of cytotoxic mediators such as, but not
limited to, IFN y, perform, and granzyme B. In some embodiments,
the improved efficacy may be shown by reduction in the signature
cytokines of the diseases, or alleviated symptoms of the disease
when the CAR expressing cells are administered to a subject. Other
cytokines that may be reduced include TGF-beta, IL-6, IL-4, IL-10,
and/or IL-13. the improved efficacy may be shown by COV-S-specific
immune cell responses, such as T cell cytotoxicity. In case of
cancer, improved efficacy may be shown by better tumor
cytotoxicity, better infiltration into the tumor, reduction of
immunosuppressive mediators, reduction in weight decrease,
reduction in ascites, reduction in tumor burden, and/or increased
lifespan. In case of autoimmune diseases, reduced responsiveness of
autoreactive cells or decrease in autoreactive T cells, B cells, or
Abs may represent improved efficacy. In some embodiments, gene
expression profiles may be also investigated to evaluate the
efficacy of the CAR.
[0528] In one aspect, the CAR expressing cells are further modified
to evade or neutralize the activity of immunosuppressive mediators,
including, but not limited to prostaglandin E2 (PGE2) and
adenosine. In some embodiments, this evasion or neutralization is
direct. In other embodiments, this evasion or neutralization is
mediated via the inhibition of protein kinase A (PKA) with one or
more binding partners, for example ezrin. In a specific embodiment,
the CAR-expressing cells further express the peptide "regulatory
subunit I anchoring disruptor" (RIAD). RIAD is thought to inhibit
the association of protein kinase A (PKA) with ezrin, which thus
prevents PKA's inhibition of TCR activation (Newick K. et al.
Cancer Immunol Res. 2016 June; 4(6):541-51).
[0529] In some embodiments, the CAR expressing cells may induce a
broad immune response, consistent with epitope spreading.
[0530] In some embodiments, the CAR expressing cells further
comprise a homing mechanism. For example, the cell may
transgenically express one or more stimulatory chemokines or
cytokines or receptors thereof. In particular embodiments, the
cells are genetically modified to express one or more stimulatory
cytokines. In certain embodiments, one or more homing mechanisms
are used to assist the inventive cells to accumulate more
effectively to the disease site. In some embodiments, the CAR
expressing cells are further modified to release inducible
cytokines upon CAR activation, e.g., to attract or activate innate
immune cells to a targeted cell (so-called fourth generation CARs
or TRUCKS). In some embodiments, CARs may co-express homing
molecules, e.g., CCR4 or CCR2b, to increase trafficking to the
disease site.
Controlling CAR Expression
[0531] In some instances, it may be advantageous to regulate the
activity of the CAR or CAR expressing cells CAR. For example,
inducing apoptosis using, e.g., a caspase fused to a dimerization
domain (see, e.g., Di et al., N Engl. J. Med. 2011 Nov. 3;
365(18):1673-1683), can be used as a safety switch in the CAR
therapy of the instant disclosure. In another example,
CAR-expressing cells can also express an inducible Caspase-9
(iCaspase-9) molecule that, upon administration of a dimerizer drug
(e.g., rimiducid (also called AP1903 (Bellicum Pharmaceuticals) or
AP20187 (Ariad)) leads to activation of the Caspase-9 and apoptosis
of the cells. The iCaspase-9 molecule contains a chemical inducer
of dimerization (CID) binding domain that mediates dimerization in
the presence of a CID. This results in inducible and selective
depletion of CAR-expressing cells. In some cases, the iCaspase-9
molecule is encoded by a nucleic acid molecule separate from the
CAR-encoding vector(s). In some cases, the iCaspase-9 molecule is
encoded by the same nucleic acid molecule as the CAR-encoding
vector. The iCaspase-9 can provide a safety switch to avoid any
toxicity of CAR-expressing cells. See, e.g., Song et al. Cancer
Gene Ther. 2008; 15(10):667-75; Clinical Trial Id. No. NCT02107963;
and Di et al. N. Engl. J. Med. 2011; 365:1673-83.
[0532] Alternative strategies for regulating the CAR therapy
include utilizing small molecules or antibodies that deactivate or
turn off CAR activity, e.g., by deleting CAR-expressing cells,
e.g., by inducing antibody dependent cell-mediated cytotoxicity
(ADCC). For example, CAR-expressing cells described herein may also
express an antigen that is recognized by molecules capable of
inducing cell death, e.g., ADCC or compliment-induced cell death.
For example, CAR expressing cells described herein may also express
a receptor capable of being targeted by an antibody or antibody
fragment. Examples of such receptors include EpCAM, VEGFR,
integrins (e.g., integrins .alpha.v.beta.3, .alpha.4,
.alpha.I3/4.beta.3, .alpha.4.beta.7, .alpha.5.beta.1,
.alpha.v.beta.3, .alpha.v), members of the TNF receptor superfamily
(e.g., TRAIL-R1, TRAIL-R2), PDGF Receptor, interferon receptor,
folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72,
IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11,
CD11a/LFA-1, CD15, CD18/ITGB2, CD19, CD20, CD22, CD23/IgE Receptor,
CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L,
CD74, CD80, CD125, CD147/basigin, CD152/CTLA-4, CD154/CD40L,
CD195/CCR5, CD319/SLAMF7, and EGFR, and truncated versions thereof
(e.g., versions preserving one or more extracellular epitopes but
lacking one or more regions within the cytoplasmic domain). For
example, CAR-expressing cells described herein may also express a
truncated epidermal growth factor receptor (EGFR) which lacks
signaling capacity but retains the epitope that is recognized by
molecules capable of inducing ADCC, e.g., cetuximab (ERBITUX.RTM.),
such that administration of cetuximab induces ADCC and subsequent
depletion of the CAR-expressing cells (see, e.g., WO2011/056894,
and Jonnalagadda et al., "Gene Ther. 2013; 20(8)853-860).
[0533] In some embodiments, the CAR cell comprises a polynucleotide
encoding a suicide polypeptide, such as for example RQR8. See,
e.g., WO2013153391A, which is hereby incorporated by reference in
its entirety. In CAR cells comprising the polynucleotide, the
suicide polypeptide may be expressed at the surface of a CAR cell.
The suicide polypeptide may also comprise a signal peptide at the
amino terminus. Another strategy includes expressing a highly
compact marker/suicide gene that combines target epitopes from both
CD32 and CD20 antigens in the CAR-expressing cells described
herein, which binds rituximab, resulting in selective depletion of
the CAR-expressing cells, e.g., by ADCC (see, e.g., Philip et al.,
Blood 2014; 124(8)1277-1287). Other methods for depleting
CAR-expressing cells include administration of CAMPATH.RTM., a
monoclonal anti-CD52 antibody that selectively binds and targets
mature lymphocytes, e.g., CAR-expressing cells, for destruction,
e.g., by inducing ADCC. In other embodiments, the CAR-expressing
cell can be selectively targeted using a CAR ligand, e.g., an
anti-idiotypic antibody. In some embodiments, the anti-idiotypic
antibody can cause effector cell activity, e.g., ADCC or ADC
activities, thereby reducing the number of CAR-expressing cells. In
other embodiments, the CAR ligand, e.g., the anti-idiotypic
antibody, can be coupled to an agent that induces cell killing,
e.g., a toxin, thereby reducing the number of CAR-expressing cells.
Alternatively, the CAR molecules themselves can be configured such
that the activity can be regulated, e.g., turned on and off, as
described below.
[0534] In some embodiments, a regulatable CAR (RCAR) where the CAR
activity can be controlled is desirable to optimize the safety and
efficacy of a CAR therapy. In some embodiments, a RCAR comprises a
set of polypeptides, typically two in the simplest embodiments, in
which the components of a standard CAR described herein, e.g., an
AB domain and an ICS domain, are partitioned on separate
polypeptides or members. In some embodiments, the set of
polypeptides include a dimerization switch that, upon the presence
of a dimerization molecule, can couple the polypeptides to one
another, e.g., can couple an AB domain to an ICS domain. Additional
description and exemplary configurations of such regulatable CARs
are provided herein and in International Publication No. WO
2015/090229, hereby incorporated by reference in its entirety.
[0535] In an aspect, an RCAR comprises two polypeptides or members:
1) an intracellular signaling member comprising an ICS domain,
e.g., a primary ICS domain described herein, and a first switch
domain; 2) an antigen binding member comprising an AB domain, e.g.,
that specifically binds a target molecule described herein, as
described herein and a second switch domain. Optionally, the RCAR
comprises a TM domain described herein. In an embodiment, a TM
domain can be disposed on the intracellular signaling member, on
the antigen binding member, or on both. Unless otherwise indicated,
when members or elements of an RCAR are described herein, the order
can be as provided, but other orders are included as well. In other
words, in an embodiment, the order is as set out in the text, but
in other embodiments, the order can be different. E.g., the order
of elements on one side of a transmembrane region can be different
from the example, e.g., the placement of a switch domain relative
to an ICS domain can be different, e.g., reversed.
[0536] In some embodiments, the CAR expressing immune cell may only
transiently express a CAR. For example, the cells may be transduced
with mRNA comprising a nucleic acid sequence encoding an inventive
CAR. In this vein, the present disclosure also includes an RNA
construct that can be directly transfected into a cell. A method
for generating mRNA for use in transfection involves in vitro
transcription (IVT) of a template with specially designed primers,
followed by polyA addition, to produce a construct containing 3'
and 5' untranslated sequences ("UTRs"), a 5' cap and/or Internal
Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a
polyA tail, typically 50-2000 bases in length (SEQ ID NO: 23193).
RNA so produced can efficiently transfect different kinds of cells.
In one embodiment, the template includes sequences for the CAR. In
an embodiment, an RNA CAR vector is transduced into a cell by
electroporation.
Target Specificity
[0537] The CAR expressing cells may further comprise one or more
CARs, in addition to the first CAR. These additional CARs may or
may not be specific for the target molecule of the first CAR. In
some embodiments, the one or more additional CARs may act as
inhibitory or activating CARs. In some aspects, the CAR of some
embodiments is the stimulatory or activating CAR; in other aspects,
it is the costimulatory CAR. In some embodiments, the cells further
include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl.
Medicine, 2013 December; 5(215): 215ra172), such as a CAR
recognizing an antigen other than the target molecule of the first
CAR, whereby an activating signal delivered through the first CAR
is diminished or inhibited by binding of the inhibitory CAR to its
ligand, e.g., to reduce off-target effects.
[0538] In some embodiments, the AB domain of the CAR is or is part
of an immunoconjugate, in which the AB domain is conjugated to one
or more heterologous molecule(s), such as, but not limited to, a
cytotoxic agent, an imaging agent, a detectable moiety, a
multimerization domain, or other heterologous molecule. Cytotoxic
agents include, but are not limited to, radioactive isotopes (e.g.,
At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and
radioactive isotopes of Lu); chemotherapeutic agents; growth
inhibitory agents; enzymes and fragments thereof such as
nucleolytic enzymes; antibiotics; toxins such as small molecule
toxins or enzymatically active toxins. In some embodiments, the AB
domain is conjugated to one or more cytotoxic agents, such as
chemotherapeutic agents or drugs, growth inhibitory agents, toxins
(e.g., protein toxins, enzymatically active toxins of bacterial,
fungal, plant, or animal origin, or fragments thereof), or
radioactive isotopes.
[0539] In some embodiments, to enhance persistence, the cells may
be further modified to overexpress pro-survival signals, reverse
anti-survival signals, overexpress Bcl-xL, overexpress hTERT, lack
Fas, or express a TGF-3 dominant negative receptor. Persistence may
also be facilitated by the administration of cytokines, e.g., IL-2,
IL-7, and IL-15.
F. B-Cell Screening and Isolation
[0540] In one embodiment, the present disclosure contemplates the
preparation and isolation of a clonal population of
antigen-specific B-cells that may be used for isolating at least
one CoV-S antigen-specific cell, which can be used to produce a
monoclonal antibody against CoV-S, which is specific to a desired
CoV-S antigen, or a nucleic acid sequence corresponding to such an
antibody. Methods of preparing and isolating said clonal population
of antigen-specific B-cells are taught, for example, in U.S. Patent
Publication No. US2007/0269868 to Carvalho-Jensen et al., the
disclosure of which is herein incorporated by reference in its
entirety. Methods of preparing and isolating said clonal population
of antigen-specific B-cells are also taught herein in the examples.
Methods of "enriching" a cell population by size or density are
known in the art. See, e.g., U.S. Pat. No. 5,627,052. These steps
can be used in addition to enriching the cell population by
antigen-specificity.
G. Methods of Producing Antibodies and Fragments Thereof
[0541] In another embodiment, the present disclosure contemplates
methods for producing anti-CoV-S antibodies and fragments thereof.
Methods of producing antibodies are well known to those of ordinary
skill in the art. For example, methods of producing chimeric
antibodies are now well known in the art (See, for example, U.S.
Pat. No. 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl.
Acad. Sci. U.S.A., 81:8651-55 (1984); Neuberger et al., Nature,
314:268-270 (1985); Boulianne, G. L. et al., Nature, 312:643-46
(1984), the disclosures of each of which are herein incorporated by
reference in their entireties).
[0542] As mentioned above, methods of producing humanized
antibodies are now well known in the art (See, for example, U.S.
Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen
et al; U.S. Pat. Nos. 5,225,539 and 6,548,640 to Winter; U.S. Pat.
Nos. 6,054,297, 6,407,213 and 6,639,055 to Carter et al; U.S. Pat.
No. 6,632,927 to Adair; Jones, P. T. et al., Nature, 321:522-525
(1986); Reichmann, L. et al., Nature, 332:323-327 (1988);
Verhoeyen, M. et al., Science, 239:1534-36 (1988), the disclosures
of each of which are herein incorporated by reference in their
entireties).
[0543] Antibody polypeptides of the disclosure having CoV-S binding
specificity may also be produced by constructing, using
conventional techniques well known to those of ordinary skill in
the art, an expression vector containing a promoter (optionally as
a component of a eukaryotic or prokaryotic operon) and a DNA
sequence encoding an antibody heavy chain in which the DNA sequence
encoding the CDRs required for antibody specificity is derived from
a non-human cell source, e.g., a rabbit or rodent B-cell source,
while the DNA sequence encoding the remaining parts of the antibody
chain is derived from a human cell source.
[0544] A second expression vector is produced using the same
conventional means well known to those of ordinary skill in the
art, said expression vector containing a promoter (optionally as a
component of a eukaryotic or prokaryotic operon) and a DNA sequence
encoding an antibody light chain in which the DNA sequence encoding
the CDRs required for antibody specificity is derived from a
non-human cell source, e.g., a rabbit or rodent B-cell source,
while the DNA sequence encoding the remaining parts of the antibody
chain is derived from a human cell source.
[0545] The expression vectors are transfected into a host cell by
convention techniques well known to those of ordinary skill in the
art to produce a transfected host cell, said transfected host cell
cultured by conventional techniques well known to those of ordinary
skill in the art to produce said antibody polypeptides.
[0546] The host cell may be co-transfected with the two expression
vectors described above, the first expression vector containing DNA
encoding a promoter (optionally as a component of a eukaryotic or
prokaryotic operon) and a light chain-derived polypeptide and the
second vector containing DNA encoding a promoter (optionally as a
component of a eukaryotic or prokaryotic operon) and a heavy
chain-derived polypeptide. The two vectors contain different
selectable markers, but preferably achieve substantially equal
expression of the heavy and light chain polypeptides.
Alternatively, a single vector may be used, the vector including
DNA encoding both the heavy and light chain polypeptides. The
coding sequences for the heavy and light chains may comprise cDNA,
genomic DNA, or both.
[0547] The host cells used to express the antibody polypeptides may
be either a bacterial cell such as E. coli, or a eukaryotic cell
such as P. pastoris. In one embodiment, a mammalian cell of a
well-defined type for this purpose, such as a myeloma cell, a CHO
cell line, a NSO cell line, or a HEK293 cell line may be used.
[0548] The general methods by which the vectors may be constructed,
transfection methods required to produce the host cell and
culturing methods required to produce the antibody polypeptides
from said host cells all include conventional techniques. Although
preferably the cell line used to produce the antibody is a
mammalian cell line, any other suitable cell line, such as a
bacterial cell line such as an E. coli-derived bacterial strain, or
a yeast cell line, may alternatively be used.
[0549] Similarly, once produced the antibody polypeptides may be
purified according to standard procedures in the art, such as for
example cross-flow filtration, ammonium sulphate precipitation,
affinity column chromatography, hydrophobic interaction
chromatography ("HIC"), and the like.
[0550] The antibody polypeptides described herein may also be used
for the design and synthesis of either peptide or non-peptide
mimetics that would be useful for the same therapeutic applications
as the antibody polypeptides of the disclosure (See, for example,
Saragobi et al., Science, 253:792-795 (1991), the contents of which
are herein incorporated by reference in its entirety).
[0551] In another embodiment, the present disclosure contemplates
methods for humanizing antibody heavy and light chains which bind
to CoV-S. Exemplary methods for humanizing antibody heavy and light
chains that may be applied to anti-CoV-S antibodies are identified
herein and are conventional in the art.
H. Screening Assays
[0552] The screening assays described here may be used to identify
high affinity anti-CoV-S Abs which may be useful in the treatment
of diseases and disorders associated with CoV-S in subjects
exhibiting symptoms of a CoV-S associated disease or disorder.
[0553] In some embodiments, the antibody is used as a diagnostic
tool. The antibody can be used to assay the amount of CoV-S present
in a sample and/or subject. As will be appreciated by one of skill
in the art, such antibodies need not be neutralizing antibodies. In
some embodiments, the diagnostic antibody is not a neutralizing
antibody. In some embodiments, the diagnostic antibody binds to a
different epitope than the neutralizing antibody binds to. In some
embodiments, the two antibodies do not compete with one
another.
[0554] In some embodiments, the antibodies disclosed herein are
used or provided in an assay kit and/or method for the detection of
CoV-S in mammalian tissues or cells in order to screen/diagnose for
a disease or disorder associated with changes in levels of CoV-S.
The kit comprises an antibody that binds CoV-S and means for
indicating the binding of the antibody with CoV-S, if present, and
optionally CoV-S protein levels. Various means for indicating the
presence of an antibody can be used. For example, fluorophores,
other molecular probes, or enzymes can be linked to the antibody
and the presence of the antibody can be observed in a variety of
ways. The method for screening for such disorders can involve the
use of the kit, or simply the use of one of the disclosed
antibodies and the determination of whether the antibody binds to
CoV-S in a sample. As will be appreciated by one of skill in the
art, high or elevated levels of CoV-S will result in larger amounts
of the antibody binding to CoV-S in the sample. Thus, degree of
antibody binding can be used to determine how much CoV-S is in a
sample. Subjects or samples with an amount of CoV-S that is greater
than a predetermined amount (e.g., an amount or range that a person
without a CoV-S-related disorder would have) can be characterized
as having a CoV-S-mediated disorder.
[0555] The present disclosure further provides for a kit for
detecting binding of an anti-CoV-S antibody of the disclosure to
CoV-S. In particular, the kit may be used to detect the presence of
CoV-S specifically reactive with an anti-CoV-S antibody or an
immunoreactive fragment thereof. The kit may also include an
antibody bound to a substrate, a secondary antibody reactive with
the antigen and a reagent for detecting a reaction of the secondary
antibody with the antigen. Such a kit may be an ELISA kit and can
comprise the substrate, primary and secondary antibodies when
appropriate, and any other necessary reagents such as detectable
moieties, enzyme substrates, and color reagents, for example as
described herein. The diagnostic kit may also be in the form of an
immunoblot kit. The diagnostic kit may also be in the form of a
chemiluminescent kit (Meso Scale Discovery, Gaithersburg, Md.). The
diagnostic kit may also be a lanthanide-based detection kit
(PerkinElmer, San Jose, Calif.).
[0556] A skilled clinician would understand that a biological
sample includes, but is not limited to, sera, plasma, urine, fecal
sample, saliva, mucous, pleural fluid, synovial fluid, and spinal
fluid.
I. Methods of Ameliorating or Reducing Symptoms of, or Treating, or
Preventing, Diseases and Disorders Associated with CoV
[0557] In another embodiment, anti-CoV-S antibodies described
herein, or antigen-binding fragments thereof, are useful for
ameliorating or reducing the symptoms of, or treating, or
preventing, diseases and disorders associated with CoV-S.
Anti-CoV-S antibodies described herein, or antigen-binding
fragments thereof, as well as combinations, can also be
administered in a therapeutically effective amount to patients in
need of treatment of diseases and disorders associated with CoV-S
in the form of a pharmaceutical composition as described in greater
detail below.
[0558] Symptoms of CoV infection may include fever, cough, runny
nose, congestion, sore throat, bronchitis, pneumonia, shortness of
breath, chest pain, headache, muscle ache, chills, fatigue,
conjunctivitis, diarrhea, loss of smell, and loss of taste.
Complications and/or diseases/disorders associated with coronavirus
infection may include, for example, bronchitis, pneumonia,
respiratory failure, acute respiratory failure, organ failure,
multi-organ system failure, pediatric inflammatory multisystem
syndrome, acute respiratory distress syndrome (a severe lung
condition that causes low oxygen in the blood and organs), blood
clots, cardiac conditions, myocardial injury, myocarditis, heart
failure, cardiac arrest, acute myocardial infarction, dysrhythmias,
venous thromboembolism, post-intensive care syndrome, shock,
anaphylactic shock, cytokine release syndrome, septic shock,
disseminated intravascular coagulation, ischemic stroke,
intracerebral hemorrhage, microangiopathic thrombosis, psychosis,
seizure, nonconvulsive status epilepticus, traumatic brain injury,
stroke, anoxic brain injury, encephalitis, posterior reversible
leukoencephalopathy, necrotizing encephalopathy, post-infectious
encephalitis, autoimmune mediated encephalitis, acute disseminated
encephalomyelitis, acute kidney injury, acute liver injury,
pancreatic injury, immune thrombocytopenia, subacute thyroiditis,
gastrointestinal complications, aspergillosis, increased
susceptibility to infection with another virus or bacteria, and/or
pregnancy-related complications. Certain diseases and conditions,
such as high blood pressure, type 1 diabetes, liver disease,
overweight, chronic lung diseases including cystic fibrosis,
pulmonary fibrosis, and asthma, compromised immune system due to
transplant, use of an immunosuppressant, or HIV infection, and
brain and nervous system condition, may increase the risk of CoV
infection-associated complications and diseases.
[0559] Also, the subject anti-CoV-S antibodies and antigen-binding
fragments may be used alone or in conjunction with other active
agents, e.g., opioids and non-opioid analgesics such as NSAIDs to
elicit analgesia. In some embodiments, aspirin and/or acetaminophen
may be taken in conjunction with the subject anti-CoV-S antibody or
antigen-binding fragment. Aspirin is another type of non-steroidal
anti-inflammatory compound.
[0560] The subject antibodies potentially optionally may be
combined with one or more of the following: (i) an antiviral drug,
optionally, remdesivir, favipiravir, darunavir, nelfinavir,
saquinavir, lopinavir, or ritonavir; (ii) an antihelminth drug,
optionally ivermectin; (iii) an antiparasitic drug, optionally
hydroxychloroquine, chloroquine, or atovaquone; (iv) antibacterial
vaccine, optionally the tuberculosis vaccine BCG; or (v) an
anti-inflammatory drug, optionally a steroid such as ciclesonide, a
TNF inhibitor (e.g., adalimumab), a TNF receptor inhibitor (e.g.,
etanercept), an IL-6 inhibitor (e.g., clazakizumab), an IL-6
receptor inhibitor (e.g., toclizumab), or metamizole; (vi) an
antihistamine drug, optionally bepotastine; (vii) an ACE inhibitor,
which is optionally moexipril; or (viii) a drug that inhibits
priming of CoV-S, optionally a serine protease inhibitor, further
optionally nafamostat. in order to increase or enhance pain
management. This may allow for such analgesic compounds to be
administered for longer duration or at reduced dosages thereby
potentially alleviating adverse side effects associated
therewith.
[0561] The subject to which the pharmaceutical formulation is
administered can be, e.g., any human or non-human animal needing
such treatment, prevention and/or amelioration, or who would
otherwise benefit from the inhibition or attenuation of
CoV-S-mediated activity. For example, the subject can be an
individual that is diagnosed with, or who is deemed to be at risk
of being afflicted by any of the aforementioned diseases or
disorders. In some instances the subject may be in an advanced
state of CoV infection, e.g., a subject who is on a ventilator. In
some instances, the subject can be one having one or more risk
factors (such as advanced age, obesity, diabetes, etc, and others
previously identified) which correlate to a poor CoV treatment or
recovery prognosis. The present disclosure further includes the use
of any of the pharmaceutical formulations disclosed herein in the
manufacture of a medicament for the treatment, prevention and/or
amelioration of any disease or disorder associated with CoV or
CoV-S activity (including any of the above-mentioned exemplary
diseases, disorders and conditions).
J. Administration
[0562] In one embodiment, the anti-CoV-S antibodies described
herein, or CoV-S binding fragments thereof, as well as combinations
of said antibodies or antigen-binding fragments thereof, are
administered to a subject at a concentration of between 0.1 mg/ml
and about any one of 0.5, 1, 5, 10, 15 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,
160, 170, 180, 190, or 200 mg/ml, +/-10% error.
[0563] In another embodiment, the anti-CoV-S antibodies and
fragments thereof described herein are administered to a subject at
a dose of between about 0.01 and 100.0 or 200.0 mg/kg of body
weight of the recipient subject. In certain embodiments, depending
on the type and severity of the CoV-S-related disease, about 1
.mu.g/kg to 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial
candidate dosage for administration to the patient, whether, for
example, by one or more separate administrations, or by continuous
infusion. In another embodiment, about 1 .mu.g/kg to 15 mg/kg
(e.g., 0.1 mg/kg-10 mg/kg) of antibody is an initial candidate
dosage for administration to the patient. A typical daily dosage
might range from about 1 .mu.g/kg to 100 mg/kg or more, depending
on several factors, e.g., the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners. However, other dosage regimens may
be useful.
[0564] For example, in addition to the relative dosages (mg/kg)
discussed herein, the subject anti-CoV-S antibodies and
antigen-binding fragments thereof can be administered to a subject
at an absolute dose (mg). Accordingly, in one embodiment, the
anti-CoV-S antibodies and antigen-binding fragments thereof
described herein are administered to a subject at a dose of between
about 1 microgram and about 1000 milligrams regardless of the route
of administration.
[0565] In a preferred embodiment, the anti-CoV-S antibodies
described herein, or anti-CoV-S antigen-binding fragments thereof,
as well as combinations of said antibodies or antigen-binding
fragments thereof, are administered to a recipient subject with a
frequency of once every twenty-six weeks or less, such as once
every sixteen weeks or less, once every eight weeks or less, once
every four weeks or less, once every two weeks or less, once every
week or less, or once daily or less.
[0566] According to preferred embodiments, the antibody containing
medicament or pharmaceutical composition is peripherally
administered to a subject via a route selected from one or more of:
orally, sublingually, buccally, topically, rectally, via
inhalation, transdermally, subcutaneously, intravenously,
intra-arterially, or intramuscularly, via intracardiac
administration, intraosseously, intradermally, intraperitoneally,
transmucosally, vaginally, intravitreally, epicutaneously,
intra-articularly, peri-articularly, or locally.
[0567] Fab fragments may be administered every two weeks or less,
every week or less, once daily or less, multiple times per day,
and/or every few hours. In one embodiment, a patient receives Fab
fragments of 0.1 mg/kg to 40 mg/kg per day given in divided doses
of 1 to 6 times a day, or in a continuous perfusion form, effective
to obtain desired results.
[0568] It is to be understood that the concentration of the
antibody or Fab administered to a given patient may be greater or
lower than the exemplary administration concentrations set forth
above.
[0569] A person of skill in the art would be able to determine an
effective dosage and frequency of administration through routine
experimentation, for example guided by the disclosure herein and
the teachings in, Goodman & Gilman's The Pharmacological Basis
of Therapeutics, Brunton, L. L. et al. editors, 11.sup.th edition,
New York, New York: McGraw-Hill (2006); Howland, R. D. et al.,
Pharmacology, Volume 864, Lippincott's illustrated reviews.,
Philadelphia, Pa.: Lippincott Williams & Wilkins (2006); and
Golan, D. E., Principles of pharmacology: the pathophysiologic
basis of drug therapy, Philadelphia, Pa.: Lippincott Williams &
Wilkins (2007).
[0570] In another embodiment, the anti-CoV-S antibodies described
herein, or CoV-S binding fragments thereof, as well as combinations
of said antibodies or antigen-binding fragments thereof, are
administered to a subject in a pharmaceutical formulation. In a
preferred embodiment, the subject is a human.
[0571] A "pharmaceutical composition" or "medicament" refers to a
chemical or biological composition suitable for administration to a
subject, preferably a mammal, more preferably a human. Such
compositions may be specifically formulated for administration via
one or more of a number of routes, including but not limited to
buccal, epicutaneous, epidural, inhalation, intraarterial,
intracardial, intracerebroventricular, intradermal, intramuscular,
intranasal, intraocular, intraperitoneal, intraspinal, intrathecal,
intravenous, oral, parenteral, rectally via an enema or
suppository, subcutaneous, subdermal, sublingual, transdermal, and
transmucosal. In addition, administration can occur by means of
injection, powder, liquid, gel, drops, or other means of
administration.
[0572] In one embodiment, the anti-CoV-S antibodies or
antigen-binding fragments thereof, as well as combinations of said
antibodies or antigen-binding fragments thereof, may be optionally
administered in combination with one or more active agents. Such
active agents include (i) an antiviral drug, optionally,
remdesivir, favipiravir, darunavir, nelfinavir, saquinavir,
lopinavir, or ritonavir; (ii) an antihelminth drug, optionally
ivermectin; (iii) an antiparasitic drug, optionally
hydroxychloroquine, chloroquine, or atovaquone; (iv) antibacterial
vaccine, optionally the tuberculosis vaccine BCG; or (v) an
anti-inflammatory drug, optionally a steroid such as ciclesonide, a
TNF inhibitor (e.g., adalimumab), a TNF receptor inhibitor (e.g.,
etanercept), an IL-6 inhibitor (e.g., clazakizumab), an IL-6
receptor inhibitor (e.g., toclizumab), or metamizole; (vi) an
antihistamine drug, optionally bepotastine; (vii) an ACE inhibitor,
optionally moexipril; or (viii) a drug that inhibits priming of
CoV-S, optionally a serine protease inhibitor, further optionally
nafamostat.
[0573] An anti-histamine can be any compound that opposes the
action of histamine or its release from cells (e.g., mast cells).
Anti-histamines include but are not limited to acrivastine,
astemizole, azatadine, azelastine, betatastine, brompheniramine,
buclizine, cetirizine, cetirizine analogues, chlorpheniramine,
clemastine, CS 560, cyproheptadine, desloratadine,
dexchlorpheniramine, ebastine, epinastine, fexofenadine, HSR 609,
hydroxyzine, levocabastine, loratadine, methscopolamine,
mizolastine, norastemizole, phenindamine, promethazine, pyrilamine,
terfenadine, and tranilast.
[0574] In CoV infection, respiratory symptoms are often exacerbated
by additional bacterial infection. Therefore, such active agents
may also be antibiotics, which include but are not limited to
amikacin, aminoglycosides, amoxicillin, ampicillin, ansamycins,
arsphenamine, azithromycin, azlocillin, aztreonam, bacitracin,
carbacephem, carbapenems, carbenicillin, cefaclor, cefadroxil,
cefalexin, cefalothin, cefalotin, cefamandole, cefazolin, cefdinir,
cefditoren, cefepime, cefixime, cefoperazone, cefotaxime,
cefoxitin, cefpodoxime, cefprozil, ceftazidime, ceftibuten,
ceftizoxime, ceftobiprole, ceftriaxone, cefuroxime, cephalosporins,
chloramphenicol, cilastatin, ciprofloxacin, clarithromycin,
clindamycin, cloxacillin, colistin, co-trimoxazole, dalfopristin,
demeclocycline, dicloxacillin, dirithromycin, doripenem,
doxycycline, enoxacin, ertapenem, erythromycin, ethambutol,
flucloxacillin, fosfomycin, furazolidone, fusidic acid,
gatifloxacin, geldanamycin, gentamicin, glycopeptides, herbimycin,
imipenem, isoniazid, kanamycin, levofloxacin, lincomycin,
linezolid, lomefloxacin, loracarbef, macrolides, mafenide,
meropenem, methicillin, metronidazole, mezlocillin, minocycline,
monobactams, moxifloxacin, mupirocin, nafcillin, neomycin,
netilmicin, nitrofurantoin, norfloxacin, ofloxacin, oxacillin,
oxytetracycline, paromomycin, penicillin, penicillins,
piperacillin, platensimycin, polymyxin B, polypeptides, prontosil,
pyrazinamide, quinolones, quinupristin, rifampicin, rifampin,
roxithromycin, spectinomycin, streptomycin, sulfacetamide,
sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole,
sulfonamides, teicoplanin, telithromycin, tetracycline,
tetracyclines, ticarcillin, tinidazole, tobramycin, trimethoprim,
trimethoprim-sulfamethoxazole, troleandomycin, trovafloxacin, and
vancomycin.
[0575] Active agents also include aldosterone, beclomethasone,
betamethasone, corticosteroids, cortisol, cortisone acetate,
deoxycorticosterone acetate, dexamethasone, fludrocortisone
acetate, glucocorticoids, hydrocortisone, methylprednisolone,
prednisolone, prednisone, steroids, and triamcinolone. Any suitable
combination of these active agents is also contemplated.
[0576] A "pharmaceutical excipient" or a "pharmaceutically
acceptable excipient" is a carrier, usually a liquid, in which an
active therapeutic agent is formulated. In one embodiment, the
active therapeutic agent is a humanized antibody described herein,
or one or more fragments thereof. The excipient generally does not
provide any pharmacological activity to the formulation, though it
may provide chemical and/or biological stability, and release
characteristics. Exemplary formulations can be found, for example,
in Remington's Pharmaceutical Sciences, Gennaro, A. editor,
19.sup.th edition, Philadelphia, Pa.: Williams and Wilkins (1995),
which is incorporated by reference.
[0577] As used herein "pharmaceutically acceptable carrier" or
"excipient" includes any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic, and
absorption delaying agents that are physiologically compatible. In
one embodiment, the carrier is suitable for parenteral
administration. Alternatively, the carrier can be suitable for
intravenous, intraperitoneal, intramuscular, or sublingual
administration. Pharmaceutically acceptable carriers include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the pharmaceutical compositions of the
disclosure is contemplated. Supplementary active compounds can also
be incorporated into the compositions.
[0578] Pharmaceutical compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
disclosure contemplates that the pharmaceutical composition is
present in lyophilized form. The composition can be formulated as a
solution, microemulsion, liposome, or other ordered structure
suitable to high drug concentration. The carrier can be a solvent
or dispersion medium containing, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol), and suitable mixtures thereof. The disclosure
further contemplates the inclusion of a stabilizer in the
pharmaceutical composition. The proper fluidity can be maintained,
for example, by the maintenance of the required particle size in
the case of dispersion and by the use of surfactants.
[0579] In many cases, it will be preferable to include isotonic
agents, for example, sugars, polyalcohols such as mannitol and
sorbitol, or sodium chloride in the composition. Absorption of the
injectable compositions can be prolonged by including an agent that
delays absorption, for example, monostearate salts and gelatin.
Moreover, the alkaline polypeptide can be formulated in a
time-release formulation, for example in a composition that
includes a slow release polymer. The active compounds can be
prepared with carriers that will protect the compound against rapid
release, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters,
polylactic acid, polylactic and polyglycolic copolymers ("PLG").
Many methods for the preparation of such formulations are known to
those skilled in the art.
[0580] For each of the recited embodiments, the compounds can be
administered by a variety of dosage forms. Any biologically
acceptable dosage form known to persons of ordinary skill in the
art, and combinations thereof, are contemplated. Examples of such
dosage forms include, without limitation, reconstitutable powders,
elixirs, liquids, solutions, suspensions, emulsions, powders,
granules, particles, microparticles, dispersible granules, cachets,
inhalants, aerosol inhalants, patches, particle inhalants,
implants, depot implants, injectables (including subcutaneous,
intramuscular, intravenous, and intradermal), infusions, and
combinations thereof.
[0581] The above description of various illustrated embodiments of
the disclosure is not intended to be exhaustive or to limit the
disclosure to the precise form disclosed. While specific
embodiments of, and examples for, the disclosure are described
herein for illustrative purposes, various equivalent modifications
are possible within the scope of the disclosure, as those skilled
in the relevant art will recognize. The teachings provided herein
of the disclosure can be applied to other purposes, other than the
examples described herein.
[0582] Certain anti-CoV-S antibody polynucleotides and polypeptides
are disclosed in the sequence listing accompanying this patent
application filing, and the disclosure of said sequence listing is
herein incorporated by reference in its entirety.
[0583] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts, manuals,
books, or other disclosures) in the Background, Detailed
Description, and Examples is herein incorporated by reference in
their entireties.
[0584] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the subject disclosure and are
not intended to limit the scope of what is regarded as the
invention. Efforts have been made to ensure accuracy with respect
to the numbers used (e.g. amounts, temperature, concentrations,
etc.), but some experimental errors and deviations should be
allowed for. Unless otherwise indicated, parts are parts by weight,
molecular weight is average molecular weight, temperature is in
degrees centigrade; and pressure is at or near atmospheric.
Examples
Example 1: Selection and Preparation of Antibodies that Selectively
Bind SARS-CoV-S and/or SARS-CoV-2-S--Using a Blood Sample from a
SARS Survivor
[0585] Studies of antibody responses to other CoVs have shown that
the S glycoprotein is the primary target for neutralizing
antibodies (nAbs) (Jiang, C. Hillyer, L. Du, Trends Immunol. 2020
Apr. 24. pii: S1471-4906(20)30087-9). Given that SARS-CoV and
SARS-CoV-2 share about 80% amino acid identity in their S proteins,
one important immunological question concerns the immunogenicity of
conserved surfaces on this antigen. Recent studies have
demonstrated that there is little to no cross-neutralizing activity
in convalescent SARS or COVID-19 sera, suggesting that conserved
antigenic sites are rarely targeted by nAbs (X. Ou et al., Nat
Commun. 2020 Mar. 27; 11(1):1620). Correspondingly, studies of
small panels of human and murine mAbs induced by SARS-CoV infection
or vaccination have shown that cross-reactivity with SARS-CoV-2 is
uncommon (D. Wrapp et al., Science. 2020 Mar. 13;
367(6483):1260-1263; Q. Wang et al., Cell. 2020 Apr. 7. pii:
S0092-8674(20)30338-X). To define conserved epitopes shared between
SARS-CoV and SARS-CoV-2, the memory B cell repertoire of a 2003
SARS survivor was mined for SARS-CoV-2 cross-reactive
antibodies.
Sample
[0586] Heparinized blood (50-100 cc) was obtained from a subject
(2003 SARS outbreak survivor) 17 years after infection with
SARS-CoV (Donor name "VRC #202367"). The sample was processed to
obtain plasma and to isolate peripheral blood-derived B cells.
Isolated cells and plasma were stored frozen in aliquots at
-80.degree. C.
Antigens and Antibodies
[0587] Production of recombinant SARS-CoV and SARS-CoV-2 spike
protein: To express the prefusion S ectodomain of SARS-CoV-2, a
gene encoding residues 1-1208 of 2019-nCoV S (GenBank: MN908947)
with proline substitutions at residues 986 and 987, a "GSAS"
substitution ("GSAS" disclosed as SEQ ID NO: 23196) at the furin
cleavage site (residues 682-685), a C-terminal T4 fibritin
trimerization motif, an HRV3C protease cleavage site, a
TwinStrepTag and an 8.times.HisTag (SEQ ID NO: 23197) was
synthesized and cloned into the mammalian expression vector paH
(DOI: 10.1126/science.abb2507). The prefusion S ectodomain of
SARS-CoV was also expressed in the same manner. Expression
construct design was based on previously described strategies for
expression of related betacoronavirus S proteins (DOI:
10.1073/pnas.1707304114 and DOI: 10.1038/s41598-018-34171-7) These
expression vector encoding SARS-CoV-2 S protein was used to
transiently transfect FreeStyle293F cells (Thermo 134 Fischer)
using polyethylenimine. Protein was purified from filtered cell
supernatants using either 135 StrepTactin resin (IBA) or Protein A
resin (Pierce) before being subjected to additional 136
purification by size-exclusion chromatography using either a
Superose 6 10/300 column (GE 137 Healthcare) or a Superdex 200
10/300 Increase column (GE Healthcare) in 2 mM Tris pH 8.0, 138 200
mM NaCl and 0.02% NaN3. The final protein preparations were stored
in phosphate-buffered saline pH 7.4 supplemented with an additional
150 mM NaCl. Small aliquots were stored at -70.degree. C. until
use.
Single B-Cell Sorting
[0588] For MBC sorting, B cells were purified using a MACS B cell
isolation kit (Miltenyi Biotec; cat #130-091-151) and subsequently
stained using anti-human CD19 (PE-Cy7), CD3 (PerCP-Cy5.5), CD8
(PerCP-Cy5.5), CD14 (PerCP-Cy5.5), CD16 (PerCP-Cy5.5), IgM (BV711),
IgD (BV421), IgA (AF488), IgG (BV605), CD27 (BV510), CD71 (APC-Cy7)
and a mixture of dual-labeled (APC and PE) SARS-CoV and/or
SARS-CoV-2 spike protein tetramers (25 nM each). Tetramers were
prepared fresh for each experiment, and B cells that showed
reactivity to the SARS-CoV and/or SARS-CoV-2 spike protein
tetramers were single cell sorted. Single cells were sorted using a
BD FACS Aria II (BD Biosciences) into 96-well PCR plates (BioRAD)
containing 20 uL/well of lysis buffer [5 uL of 5.times. first
strand cDNA buffer (Invitrogen), 0.625 uL of NP-40 (New England
Biolabs), 0.25 uL RNaseOUT (Invitrogen), 1.25 uL dithiothreitol
(Invitrogen), and 12.6 uL dH2O]. Plates were immediately stored at
-80.degree. C. Flow cytometry data were analyzed using FlowJo
software.
[0589] As shown in FIG. 6A, flow cytometric analysis revealed that
approximately 0.15% of class-switched memory B cells were
SARS-CoV-2 S-reactive, which was about 3-fold over background
staining observed with a SARS-CoV-naive donor sample. Notably, the
frequency of antigen-specific memory B cells was higher than
expected, given the long interval between infection and blood draw
(17 years) and previous studies showing that SARS-CoV-specific
memory B cells typically wane to undetectable levels after only 6
years (Tang F. et al., J Immunol. 2011 Jun. 15;
186(12):7264-8).
Amplification and Cloning of Antibody Variable Genes
[0590] Antibody variable genes (IgH, IgK, and IgL) were amplified
by reverse transcription PCR and nested PCRs using cocktails of
IgG- and IgM-specific primers, as described previously (Tiller et
al, J Immunol 2008). The primers used in the second round of PCR
contained 40 base pairs of 5' and 3' homology to the digested
expression vectors, which allowed for cloning by homologous
recombination into S. cerevisiae. The lithium acetate method for
chemical transformation was used to clone the PCR products into S.
cerevisiae (Gietz and Schiestl, Nat Protoc 2007). 10 uL of
unpurified heavy chain and light chain PCR product and 200 ng of
the digested expression vectors were used per transformation
reaction. Following transformation, individual yeast colonies were
picked for sequencing and characterization. Cognate antibody heavy-
and light-chain pairs were rescued from 315 individual
SARS-CoV-2-reactive B cells and cloned. The sequences, the germline
origins, and isotypes were determined. The number of nucleic acid
substitutions relative to the germline sequences and the number of
amino acid alterations relative to the germline-encoded sequences
were also analyzed.
[0591] The results are shown in FIGS. 3-6. Sequence analysis
revealed that about half of the clones were members of expanded
clonal lineages (the combination of VH1-69 and VK2-30 (shown in
blue), or other combinations (shown in dark gray) such as those of
VH1-69 and a VL that is not VK2-30), whereas the other half were
unique (FIG. 6C). This result contrasts with many studies of other
primary viral infections, which have shown a limited degree of
clonal expansion within the antigen-specific memory B cell
repertoire (Rogers T. F. et al., Sci Immunol. 2017 Aug. 18; 2(14);
Goodwin E., et al., Immunity. 2018 Feb. 20; 48(2); Bornholdt Z. A.,
et al., Science. 2016 Mar. 4; 351(6277):1078-83; Wec A. Z., et al.,
Proc Natl Acad Sci USA. 2020 Mar. 24; 117(12):6675-6685). In
addition, many clonally unrelated antibodies displayed convergent
VH1-69/VK2-30 germline gene pairing (FIG. 6C). As expected, almost
all of the isolated antibodies were somatically mutated, with
members of clonally expanded lineages showing higher levels of
somatic hypermutation (SHM) compared to unique clones (FIG. 6D).
Finally, index sorting analysis indicated that 33% and 66% of
binding antibodies originated from IgA+ and IgG+ memory B cells,
respectively (FIG. 6E). These results suggest that SARS-CoV
infection elicited a high frequency of long-lived, cross-reactive
memory B cells (MBCs) in this donor.
Expression and Purification of IgGs and Fab Fragments
[0592] IgGs were expressed in S. cerevisiae cultures grown in
24-well plates, as described previously (Bornholdt et al, Science
2016, PMID:26912366). After 6 days, the cultures were harvested by
centrifugation and IgGs were purified by protein A-affinity
chromatography. The bound antibodies were eluted with 200 mM acetic
acid/50 mM NaCl (pH 3.5) into 1/8th volume 2 M Hepes (pH 8.0), and
buffer-exchanged into PBS (pH 7.0). Of the 315 cloned antibodies,
202 bound to SARS-CoV-2 S in preliminary binding screens (FIG.
6B).
[0593] The antibody CR3022 was cloned into S. cerevisiae using
recombinational cloning. The variable region sequences of CR3022
were synthesized as gBlock fragments (IDT) with homologous
overhangs which were cloned and expressed as described above.
[0594] Fab fragments were generated by digesting the IgGs with
papain for 2 h at 30.degree. C. The digestion was terminated by the
addition of iodoacetamide, and the Fab and Fc mixtures were passed
over Protein A agarose to remove Fc fragments and undigested IgG.
The flowthrough of the Protein A resin was then passed over
CaptureSelect.TM. IgG-CH1 affinity resin (ThermoFischer
Scientific), and eluted with 200 mM acetic acid/50 mM NaCl pH 3.5
into 1/8th volume 2M Hepes pH 8.0. Fab fragments then were
buffer-exchanged into PBS pH 7.0.
Example 2: Kinetics of Binding Measurements (S Protein of SARS-CoV
and SARS-CoV-2)
[0595] Next, the apparent binding affinities (K.sub.D.sup.APPs) of
the antibodies to prefusion-stabilized SARS-CoV and SARS-CoV-2 S
proteins was measured (D. Wrapp et al., Cryo-EM structure of the
2019-nCoV spike in the prefusion conformation. Science 367,
1260-1263 (2020)).
Bio-Layer Interferometry Kinetic Measurements (BLI):
[0596] For monovalent apparent KD determination, IgG binding to
recombinant SARS CoV or SARS-CoV-2 spike protein antigen was
measured by biolayer interferometry (BLI) using a FortdBio Octet
HTX instrument (Molecular Devices). The IgGs were captured (1.5 nm)
to anti-human IgG capture (AHC) biosensors Molecular Devices) and
allowed to stand in PBSF (PBS with 0.1% w/v BSA) for a minimum of
30 min. After a short (60 s) baseline step in PBSF, the IgG-loaded
biosensor tips were exposed (180 s, 1000 rpm of orbital shaking) to
SARS CoV or SARS-CoV-2 spike protein (100 nM in PBSF) and then
dipped (180 s, 1000 rpm of orbital shaking) into PBSF to measure
any dissociation of the antigen from the biosensor tip surface.
Data for which binding responses were >0.1 nm were aligned,
inter-step corrected (to the association step) and fit to a 1:1
binding model using the FortdBio Data Analysis Software, version
11.1.
[0597] For bivalent apparent KD determination, IgG binding to the
SARS-CoV-2 NTD and RBD was measured by BLI using a FortdBio Octet
HTX instrument (Molecular Devices). Recombinant biotinylated
antigens were immobilized on streptavidin biosensors (Molecular
Devices) and allowed to stand in PBSF (PBS with 0.1% w/v BSA) for a
minimum of 30 min. After a short (60 s) baseline step in PBSF, the
antigen-loaded biosensor tips were exposed (180 s, 1000 rpm of
orbital shaking) to the IgGs (100 nM in PBSF) and then dipped (180
s, 1000 rpm of orbital shaking) into PBSF to measure any
dissociation of the IgGs from the biosensor tip surface. Data for
which binding responses were >0.1 nm were aligned, interstep
corrected (to the association step) and fit to a 1:1 binding model
using the FortdBio Data Analysis Software, version 11.1.
[0598] The SARS-CoV-2 S1 subunit was purchased from Acro Biosystems
(Cat #S1N-C52H3) and SARS-CoV-2 S2 subunit was purchased from Sino
Biological (Cat #S2N-C52H5).
[0599] The binding kinetics for all tested antibodies are provided
in FIG. 7. Binding affinities (KD [M]) for SARS-CoV-S and
SARS-CoV-2-S for each antibody are shown in a dot plot in FIG. 14A.
While the majority of mAbs (153 out of 202) showed binding to both
SARS-CoV-2 and SARS-CoV S, a subset of mAbs appeared to be
SARS-CoV-2 S-specific. This result was unexpected given the mAbs
were isolated from a donor who had been infected with SARS-CoV and
may relate to differences between the infecting SARS-CoV strain and
the recombinant SARS-CoV S protein (Tor2) used for the binding
studies. Alternatively, this result may be due to inherent
differences in the stability or antigenicity of recombinant
prefusion-stabilized SARS-CoV and SARS-CoV-2 S proteins. Indeed,
about 30% of antibodies that failed to bind recombinant SARS-CoV S
displayed reactivity with SARS-CoV S expressed on the surface of
transfected cells, providing some evidence for differences in the
antigenicity of recombinant and cell-expressed forms of S (FIG.
20F). Interestingly, most of the highly mutated and clonally
expanded antibodies bound to both SARS-CoV and SARS-CoV-2 S with
K.sub.D.sup.APP>10 nM (FIG. 14B).
Example 3: Kinetics of Binding Measurements (S Protein of
Circulating/Seasonal CoV Species)
[0600] Next, it was determined whether these antibodies originated
from pre-existing MBCs that were induced by prior exposures to
naturally circulating HCoVs, which share up to 32% amino acid
identity with SARS-CoV and SARS-CoV-2 in their S proteins.
Accordingly, binding affinities to the S protein of HCoV-229E,
HCoV-HKU1, HCoV-NL63, and HCoV-OK43 were measured and analyzed in a
similar manner as in Example 2. The S proteins from 229E, NL63 and
OC43 were purchased from Sino Biological (Cat. #40605-V08B,
40604-V08B and 40607-V08B).
[0601] Results are shown in FIGS. 22-25.
[0602] Based on the results from Examples 2 and 3, the binding
specificity/the broadness of the reactivity for each antibody was
determined (FIG. 12). Antibodies with the binding response value
(nM) of 0.1 or higher for a given target were considered as binders
and shown as "Yes". Also antibodies with a binding response value
such as 0.099 or 0.098 that can be round up to 0.1 were also
identified as binders and shown as "Yes". Antibodies with the
binding response value (nM) of <0.1 for a given target were
considered to be non-binders and shown as "No". Antibodies that are
"Yes" for SARS-CoV-S and/or SARS-CoV-2-S but "No" for all of the S
proteins of HCoV-229E, HCoV-HKU1, HCoV-NL63, and HCoV-OK43 were
classified as "SARS 1-2 specific". Antibodies that are "Yes" for
SARS-CoV-S and/or SARS-CoV-2-S and "Yes" for at least one of the S
proteins of HCoV-229E, HCoV-HKU1, HCoV-NL63, and HCoV-OK43 were
classified as "Broad" (binders).
[0603] Polyspecificity (also referred to as polyreactivity) is a
highly undesirable property that has been linked to poor antibody
pharmacokinetics (Wu et al., J Mol Biol 368:652-665, 2007; Hotzel
et al., 2012, MAbs 4(6):753-760) and, thus, potentially to poor
developability. Antibodies can be detected as possessing decreased
or increased developability by virtue of their level of interaction
with polyspecificity reagent (PSR). See WO2014/179363. Antibodies
displaying increased interaction with PSR are referred to as
"polyspecific" polypeptides, with poor(er) developability. SARS2
antibodies selected or identified as possessing enhanced
developability based on low polyspecificity score are considered
"developable".
[0604] A poly specificity of each antibody was measured as
described previously (L. Shehata et al., Affinity Maturation
Enhances Antibody Specificity but Compromises Conformational
Stability. Cell reports 28, 3300-3308 e3304 (2019)). Briefly,
soluble membrane protein (SMP) and soluble cytosolic protein (SCP)
fractions obtained from Chinese hamster ovary (CHO) cells were
biotinylated using NHS-LC-Biotin (Thermo Fisher Scientific Cat
#21336). IgGs presented on the surface of yeast were incubated with
1:10 diluted biotinylated CHO cell preparations on ice for 20
minutes. Cells were then washed twice with ice-cold PBS containing
0.1% BSA (PBSF) and incubated in 50 .mu.L of a secondary labelling
mix containing ExtrAvidin-R-PE (Sigma-Aldrich), anti-human LC-FITC
(Southern Biotech) and propidium iodide) for 15 minutes. The cells
were washed twice with PBSF and resuspended in PBSF to be run on a
FACSCanto II (BD Biosciences). The mean fluorescence intensity of
binding was normalized using control antibodies that display low,
medium or high polyspecificity to assess the non-specific binding.
The results are shown in the tables in FIGS. 13A-13C and the graph
in FIG. 13D in comparison with clinical antibodies.
[0605] The polyspecificity score can be useful, as one or one of
many metrics, in ranking the antibodies based on developability.
For example, the antibodies can be ranked as clean (below 0.11),
low (below 0.33), medium (below 0.66), and high polyspecificity
(above 0.66) as in FIG. 13D. In FIGS. 13A-13C, the antibodies were
categorized as A when the Score is below 0.10. and as B when the
Score is 0.10 or higher. As visualized in FIG. 13D, the vast
majority of cross-reactive (i.e., bind to SARS-CoV and SARS-CoV-2)
antibodies lacked polyspecificity, demonstrating that the observed
broad binding activity is not due to non-specific
cross-reactivity.
[0606] The results from Examples 2 and 3 are further analyzed and
summarized in the graphs in FIG. 14. Over half of the
cross-reactive (i.e., bind to SARS-CoV-S and SARS-CoV-2-S)
antibodies (89/153) bound with KDApp>10 nM to both SARS-CoV and
SARS-CoV-2 S (FIGS. 14A and 14B). As shown in FIG. 14B, over 80% of
such SARS-CoV/SARS-CoV-2 cross-reactive antibodies having KDApp
values of >10 nM showed reactivity with one or more of the
circulating CoVs, a subset of which displayed preferential binding
to circulating CoV S proteins, suggesting that these B cells may
have been initially induced by seasonal CoV exposure and
subsequently activated and expanded by SARS-CoV infection.
Alternatively, circulating HCoV infections experienced by this
donor after the SARS-CoV infection may have expanded this
cross-reactive memory B cell (MBC) pool. Consistent with this
hypothesis, the broadly cross-reactive antibodies showed
significantly higher levels of somatic hypermutation ("SHM") and
clonal expansion compared to the antibodies that only recognized
SARS-CoV and SARS-CoV-2, consistent with a recall memory B cell
response (FIGS. 14C and 14D). Notably, 72% of the broad binding
mAbs utilized VH1-69/VK2-30 germline gene pairing, suggesting
germline-mediated recognition of a common antigenic site (FIGS. 14B
and 14H). Index sorting analysis revealed that the majority of
broad binding antibodies were derived from IgA memory B cells,
indicating a mucosal origin, whereas most of the
SARS-CoV/SARS-CoV-2 cross-reactive antibodies originated from IgG
memory B cells (FIG. 14E).
[0607] Although SARS-CoV-2 S-reactive antibodies were identified
from all three naive donors, they displayed lower levels of SHM,
clonal expansion, and binding affinities for both SARS-CoV and
SARS-CoV-2 S compared to the cross-reactive antibodies identified
from the convalescent SARS donor (FIGS. 14F and 14G). Altogether,
these results suggest that SARS-CoV infection likely led to the
activation and expansion of pre-existing cross-reactive memory B
cells induced by circulating CoVs or vice versa.
[0608] To investigate whether the above results were due to an
original antigenic sin ("OAS") phenomenon, or instead, as was
hoped, due to avid binding of circulating HCoV-specific B cell
receptors to the SARS-CoV-2 S tetramers used for cell sorting,
whether similarly broadly binding antibodies were also present in
sarbecovirus-naive donors that had been exposed to endemic HCoVs by
ELISA was assessed. Peripheral blood mononuclear cell (PBMC)
samples were obtained from three healthy adult donors with
serological evidence of circulating HCoV exposure and no history of
SARS-CoV or SARS-CoV-2 infection and stained the corresponding B
cells with a fluorescently labeled SARS-CoV-2 S probe (FIGS. 141
and 14J). Flow cytometric analysis revealed that between 0.06-0.1%
of total B cells in the three naive donors displayed SARS-CoV-2
reactivity (FIG. 14K). Over 350 SARS-CoV-2-reactive MBCs were
sorted and amplified by single-cell RT-PCR, and 141 VH/VL pairs
were cloned and expressed as full-length IgGs. Although a limited
number of SARS-CoV-2 S binding antibodies were identified from all
three naive donors, they displayed significantly lower levels of
SHM, clonal expansion, and binding affinities for both SARS-CoV and
SARS-CoV-2 S compared to the cross-reactive antibodies identified
from the convalescent SARS donor (FIGS. 14F and 14G and FIG. 14L).
Altogether, these results suggest that SARS-CoV infection likely
led to the activation and expansion of pre-existing cross-reactive
MBCs induced by circulating HCoV exposure.
Example 4: Epitope Mapping
[0609] Epitope mapping was performed using different subunit and
domain constructs of SARS-CoV-2-S using the Forte bio kit and
yeast-based competition assays. Due to the inherent technical
challenges associated with measuring binding of certain antibodies
to monomeric proteins, these studies were restricted to the 65
binders with K.sub.D.sup.Apps<10 nM to SARS-CoV-2 (FIG. 7).
[0610] The domain/subunit used for the binding studies were the S1
subunit, S2 subunit, the RBD, and NTD and for the binding to S1 and
NTD, monovalent binding and bivalent ("AVID") binding were both
measured.
[0611] The results in FIGS. 15-18 are further summarized in FIG. 19
and FIG. 21F. Again the antibodies with a binding response value of
0.1 or higher were considered as binders (shown as "Yes"). Also
again the antibodies with a binding response value such as 0.099 or
0.098 that can be round up to 0.1 were also considered as binders
and shown as "Yes". Antibodies with a binding response value of
<0.1 were considered to be non-binders (shown as "Non").
[0612] As shown in FIGS. 15-19 and 21F, 75% of the antibodies
recognized epitopes within S1, whereas the remaining 25% bound to
epitopes within S2, and among 49 S1-directed antibodies 21 (43%)
and 28 (57%) recognized the RBD and NTD, respectively.
[0613] For the competition studies, the competition of each
antibody with recombinant human ACE2 ("hACE2") and with a
commercial antibody CR3022, which targets a conserved epitope
outside of the receptor binding site, was tested (M. Yuan et al., A
highly conserved cryptic epitope in the receptor-binding domains of
SARS-CoV-2 and SARS-CoV. Science, (2020)).
[0614] The results are shown in FIGS. 21A-21E. As shown six of the
antibodies competed only with hACE2, three competed only with
CR3022, four competed with both hACE2 and CR3022, and seven did not
compete with either hACE2 or CR3022.
Example 5: Cell Binding Assays
[0615] HEK-293 cells were transiently transfected with a plasmid
encoding the S protein of SARS-CoV or of SARS-CoV-2 or with an
empty plasmid, using the transfection reagent PEI. Antibodies were
incubated with the HEK-293 cells engineered to express SARS-CoV-2-S
or the HEK-293 cells transfected with an empty plasmid ("wild type"
cells or "WT" cells), and the binding was measured. The fold
binding to the S protein-expressing cells over the WT cells and the
EC50 [nM] values were calculated. The results for SARS-CoV-2-S cell
binding are shown in FIGS. 20A-20E and the results for SARS-CoV-S
cell binding are shown in FIG. 20F.
Example 6: Micro-Titer Neutralization Assays
[0616] To evaluate the neutralization potency of the
SARS-CoV/SARS-CoV-2 cross-reactive antibodies, neutralization
assays were performed using both VSV- and murine leukemia virus
(MLV)-based pseudotype systems as well as authentic viruses of
SARS-CoV and SARS-CoV-2. Due to the large number of antibodies,
initial neutralization screening was performed in an authentic
virus assay using a single concentration of purified IgG.
6-1: Authentic SARS-CoV and SARS-CoV-2 Neutralization Assay
[0617] Vero E6 cells were inoculated with SARS-CoV-2/MT020880.1 or
SARS-CoV/Urbani at an MOI=0.01 and incubated at 37.degree. C./5%
CO2/80% humidity. At 50 hours post-infection, cells were frozen at
-80.degree. C. for 1 hour, allowed to thaw at room temperature, and
supernatants were collected and clarified by centrifugation at
.about.2500.times.g for 10 minutes. Clarified supernatant was
aliquoted and stored at -80.degree. C. Sequencing data for the
SARS-CoV-2 virus stock indicated a single mutation in the spike
glycoprotein (H655 to Y) relative to Washington state isolate
MT020880.1. Sequencing data is not available for the SARS-CoV
stock.
[0618] A pre-titrated amount of authentic SARS-CoV-2/MT020880.1 or
SARS-CoV/Urbani, at final multiplicity of infection of 0.4 and
0.2., respectively, was incubated with serial dilutions of
monoclonal antibodies for 1 h at room temperature. The
antibody-virus mixture was applied to monolayers of Vero-E6 cells
in a 96-well plate and incubated for 1 hour at 37.degree. C. in a
humidified incubator. Infection media was then removed and cells
were washed once with 1.times.PBS, followed by addition of fresh
cell culture media. Culture media was removed 24 hours post
infection and cells were washed once with 1.times.PBS. PBS was
removed and plates were submerged in formalin fixing solution, then
permeabilized with 0.2% Triton-X for 10 minutes at room temp and
treated with blocking solution. Infected cells were detected using
a cross-reactive primary detection antibody recognizing SARS-CoV
and SARS-CoV-2 nucleocapsid protein (Sino Biological) 5 following
staining with secondary detection antibody (goat a rabbit)
conjugated to AlexaFluor 488. Infected cells were enumerated using
Operetta high content imaging instrument and data analysis was
performed using the Harmony software (Perkin Elmer).
6-2: rVSV-SARS-CoV-2 Neutralization Assay
[0619] A recombinant vesicular stomatitis virus (rVSV) expressing
an eGFP reporter and encoding the SARS-CoV-2 spike protein gene
(SEQ ID NO: 6) in place of its native glycoprotein is generated by
plasmid-based rescue as described previously (Whelan et al., 1995;
PMID: 7667300 and Kleinfelter et al., 2015; PMID: 26126854). The
identity of the generated virus is confirmed by Sanger sequencing
of the spike protein encoding gene after RT-PCR amplification.
[0620] A pre-titrated amount of rVSV-SARS2 S virus is incubated
with serial dilutions of monoclonal antibodies for 1 hr at room
temperature. The antibody-virus mixture is applied to monolayers of
Huh7.5.1 cells in a 384-well plate. Following overnight incubation,
eGFP-positive virus-infected cells are enumerated using a
Cytation-5 imager (Biotek) and analyzed with the onboard Gen5
software.
6-3: SARS-CoV-MLV and SARS-CoV-2-MLV Pseudo Viral Particle
Neutralization Assay
[0621] To generate pseudovirus, SARS-CoV (AAP13567) and SARS-CoV2
(NC_045512) spike genes (codon optimized for mammalian expression)
were synthesized (IDT) with 18 and 28 amino acid c-terminal
deletions respectively and cloned into pCDNA3.3 (ThermoFisher). A
luciferase reporter gene plasmid (addgene #18760) was modified to
replace the IRES with a CMV promoter. These plasmids along with MLV
gag/pol (addgene #14887) were purified using Endo-Free plasmid maxi
kits (Qiagen, #12362). Under sterile conditions, these plasmids
were co-transfected into HEK293T cells using Lipofectamine 2000
(ThermoFischer Scientific, 11668019) according to the
manufacturer's directions to produce single-round of infection
competent pseudoviruses. 0.5 ug SARS-CoV-2 or 1 ug SARS-CoV-1 S, 2
ug gag/pol and 2 ug MLV luciferase was used per well of a 6-well
plate. The media was changed 16 hours post transfection. The
supernatant containing SARS S-pseudotyped viral particles was
collected 48 h post transfection, aliquoted, and frozen at
-80.degree. C. for the neutralization assay. n
[0622] SARS-CoV-1 and SARS-CoV-2 neutralization by monoclonal
antibodies was assessed using a Murine Leukemia Pseudovirus assay
described in (ref). Briefly, SARS Spike (S) protein pseudotyped
Murine Leukemia Virus (MLV) containing a firefly luciferase
reporter gene was titrated with various mAbs and subsequently added
to hACE2-expressing Hela cells where infection was monitored using
a luminescence assay.
[0623] The neutralization assay was performed as previously
described with minor modifications (PMID:24291345). In sterile
white 96-well half-area plates (Corning, #3688), 25 .mu.l of SARS1
or SARS2 pseudotyped MLV vector was immediately mixed with 25 .mu.l
of serially diluted (5.times. using media) mAbs and incubated for
one hour at 37.degree. C. to allow for antibody neutralization of
the pseudotyped virus. 10,000 HeLa-hACE2 cells/well (in 50 ul of
media containing 20 g/ml Dextran) were directly added to the
antibody virus mixture. Plates were incubated at 37.degree. C. for
42 to 48 h. Following the infection, HeLa-hACE2 cells were lysed
using 1.times. luciferase lysis buffer (25 mM Gly-Gly pH 7.8, 15 mM
MgSO4, 4 mM EGTA, 1% Triton X-100). Luciferase intensity was then
read on a Luminometer with luciferase substrate according to the
manufacturer's instructions (Promega, PR-E2620). Percentage of
neutralization was calculated using the following equation.
100 * ( 1 - RULs .times. .times. of .times. .times. sample -
Average .times. .times. RULs .times. .times. of .times. .times.
Background Average .times. .times. of .times. .times. RULs .times.
.times. of .times. .times. Virus .times. .times. only .times.
.times. contrl - Average .times. .times. RULs .times. .times. of
.times. .times. Background ) ##EQU00001##
[0624] The results from 6-1 through 6-3 are shown in FIG. 22, FIGS.
23F and 23G show neutralization results on selected antibodies.
[0625] Also, the authentic virus neutralization results were
analyzed together with the results from the epitope analysis from
Example 4. As shown in FIGS. 23A and 23B, nine out of 202
antibodies showed >40% neutralization at 100 nM using the
authentic virus system, and eight of the nine antibodies bind to
the RBD (FIG. 23A) and compete with hACE2 (FIG. 23B) and one of the
nine antibodies bind to the NTD (FIG. 23A). Of the nine, seven
antibodies showed >90% neutralization (FIGS. 22 and 23A). All
seven of these neutralizing antibodies bind to the RBD and compete
with hACE2 (FIGS. 22, 23A, and 23B). Of the eight RBD-directed
antibodies, four bound to epitopes overlapping both hACE2 and
CR3022 (FIG. 30D) and the other four recognized epitopes only
overlapping that of hACE2 (FIG. 23C), suggesting the existence of
at least two overlapping but distinct neutralizing epitopes within
the RBD. None of the antibodies that bind to the RBD, but which do
not compete with hACE2, exhibited neutralization at 100 nM (FIG.
23C).
[0626] Titration studies further demonstrated that the nAbs
displayed IC.sub.50 s ranging from 0.6-20 gg/ml against authentic
SARS-CoV-2, and significantly, none of the antibodies left an
un-neutralized fraction of virus (FIG. 23D).
[0627] Interestingly, little to no correlation was observed between
binding affinity for cell surface S and neutralizing activity (FIG.
23E). For example, all of the S2-directed antibodies and a subset
of NTD-directed antibodies bound with high affinity to both
recombinant and cell surface S, but none of these antibodies
displayed significant neutralizing activity. Similarly, the
RBD-directed antibodies targeting epitopes outside of the hACE2
binding site showed little to no neutralizing activity, despite
binding with similar or even higher affinity to cell surface S
compared to the hACE2 competitor antibodies.
[0628] The neutralization data from using different viral systems
for the nine antibodies that showed >40% neutralization of
authentic viruses are further summarized in FIG. 25. The IC50
values and KD values of seven antibodies that showed neutralization
using two or more viral/pseudoviral systems in FIG. 25 are
summarized in FIG. 26. Based on their binding and neutralization
properties these antibodies were selected as being ideal candidates
for further affinity maturation.
[0629] Neutralization of MLV pseudotype SARS-CoV and SARS-CoV-2 by
eight antibodies are shown in FIG. 23F. All eight antibodies
neutralized both SARS-CoV and SARS-CoV-2 pseudo-MLV. Neutralization
of authentic SARS-CoV and SARS-CoV-2 by seven antibodies are shown
in FIG. 23G. All seven antibodies neutralized both SARS-CoV and
SARS-CoV-2. ADI-55688, ADI-55689, ADI-55993, and ADI-56046
(arrowed) were further subjected to affinity maturation in Example
12. FIG. 24 shows the neutralization results obtained using the
pseudo system (MLV or rVSV) corelate well with the results obtained
using the authentic viruses.
Example 7: Cluster Analysis of Antibodies Based on VH CDR3
[0630] A clustering analysis on antibodies assigned with BD Index
Numbers 1-71 was performed based on the VH CDR3 amino acid
sequences using the parameters below:
[0631] 1) Levenshtein distance <=3
[0632] 2) Aggressive clustering to merge clusters
[0633] 3) Reduced alphabet: (AST), (G), (P), (FWY), (ILVMC), (DE),
(NQH), (RK)
[0634] FIGS. 27A-27C provide clusters based on VH CDR3. FIGS.
28A-28B provides sample plots for the clusters containing more than
2 antibodies (Clusters 1 through 5).
[0635] Antibodies that are classified in Cluster 1 are ADI-55702,
ADI-55704, ADI-55706, ADI-55723, ADI-55725, ADI-55726, ADI-55728,
ADI-55731, ADI-55739, ADI-55741, ADI-55743, ADI-55745, and
ADI-55748. Antibodies that are classified in Cluster 2 are
ADI-55700, ADI-55705, ADI-55712, ADI-55717, ADI-55736, ADI-55742,
and ADI-55747. Antibodies that are classified in Cluster 3 are
ADI-55695, ADI-55698, and ADI-55714. Antibodies that are classified
in Cluster 4 are ADI-55688, ADI-55691, and ADI-55693. Antibodies
that are classified in Cluster 5 are ADI-55708, ADI-55709, and
ADI-55719. Among these antibodies, ADI-55700, ADI-55688, ADI-55708,
ADI-55709, and ADI-55719 are those that were determined to be
cross-reactive. Particularly, the results from Example 2 and
Example 6 were compared and it was discovered that Cluster 5
antibodies, ADI-55708, ADI-55709, and ADI-55719, are all
cross-reactive antibodies, and interestingly, share the identical
VH CDR3 sequence, which is ARGSLSREYDFLTAPQNGPWFDS (SEQ ID NO:
2108, 2208, or 3208) suggesting that there is a structure-feature
relationship between this particular VH CDR3 sequence and the
ability of the antibodies containing this VH CDR3 polypeptide to
cross-react with SARS-CoV-S and SARS-CoV-2-S. Moreover, since these
three antibodies also share the same VH CDR1 as well, it is further
possible that the VH CDR1 may also contribute to the
structure-feature relationship, i.e., the ability to cross-react
with SARS-CoV-S and SARS-CoV-2-S.
Example 8: FRNT Assay
[0636] For SARS CoV or SARS-CoV-2 spike protein binding ELISAs,
96-well plates (Corning; Cat #3690) were coated with 5 .mu.g/ml of
SARS CoV or SARS-CoV-2 spike protein diluted in PBS and incubated
overnight at 4.degree. C. Wells were washed and then blocked with
5% non-fat dried milk (NFDM) in PBS for 1 hour at 37.degree. C.
Wells were washed 3 times with PBS and serial dilutions of human
plasm in 5% NFDM-PBS were added and incubated for 1 hour at
37.degree. C. Plates were then washed 3 times with PBS and
secondary cross-adsorbed anti-human IgG-HRP (Thermo Fisher
Scientific; cat #31413) or anti-human-IgM (Sigma Aldrich; cat
#AP114P) detection antibodies were added at 1:8000 dilution in 5%
NFDM-PBS for 1 hour at 37.degree. C. After washing 3 times with PBS
detection reagent was added per manufacturer recommendations
(Thermo Scientific; Cat #34029) and absorbance was measures at 450
nM wavelength using a Spectramax microplate Reader (Molecular
Devices).
Example 9: In Vivo Efficacy
[0637] Therapeutic and/or Prophylactic Efficacy
[0638] Test animals, preferably mammals such as mice, rats,
rabbits, pigs, or monkeys, will be split in multiple groups. An
antibody or fragment thereof of the present disclosure will be
administered to at least one group. At least one group will not
receive such antibody or fragment thereof. The animals will then be
infected with coronavirus (SARS-CoV, SARS-CoV-2, MERS-CoV, or a
seasonal CoV). Alternatively, the antibody or fragment thereof may
be administered before the infection with CoV. The antibody or
fragment thereof may be given intravenously, intraperitoneally,
intranasally, or via any other appropriate route.
[0639] The body weight of each animal will be monitored. Symptoms
such as fever or mobility may also be monitored. Periodically,
samples such as serum will be harvested and the viral load will be
measured. Survival will be tracked. Animals may be sacrificed based
on the pre-determined cutoff value of the body weight and/or
viremia and/or the behavior and/or symptom(s).
Example 10: Prediction of CoV Vaccine Efficacy
[0640] Some of the antibodies of the present disclosure neutralize
CoV ("nAb"). Some of the antibodies do not neutralize CoV
("non-nAb"). The use of these antibodies for predicting whether a
given CoV vaccine composition will elicit protective immune
responses, such as neutralizing antibodies when the vaccine is
administered to a subject, is envisioned. This will be achieved by
quantifying the binding of the vaccine composition with nAb and/or
non-Ab and determining whether the composition binds to, binds
sufficiently to, or binds more to nAbs.
[0641] Different vaccine compositions may be compared, perhaps for
the screening purposes, to determine which vaccine is predicted to
elicit more or better protective immune responses such as
neutralizing antibodies. For example, this may be done by preparing
a panel of different nAb(s) and non-nAb(s) and test which vaccine
composition binds more to the nAb(s) than to the non-nAb(s).
Vaccine compositions that bind more to the nAb(s) would be
predicted to be more effective in eliciting protective immune
responses.
[0642] Furthermore, CoV-S is a highly glycosylated protein, and
therefore the glycosylation can vary depending on how the S protein
is prepared. For example, the type of cells, the expression method
and/or conditions, purification method and/or conditions, or even
storage can affect the glycosylation. All these factors can affect
whether a putative vaccine contains at least one epitope to which a
vaccinated individual will elicit an immune response against which
will be sufficient in order to mount a protective immunity. It is
anticipated that the antibodies may aid in determining whether a
putative vaccine expresses a CoV-S protein having the appropriate
glycosylation in order to elicit neutralizing antibodies.
[0643] Broadly protective vaccines against known and pre-emergent
coronaviruses are urgently needed. When the purpose of vaccine is
to induce immune responses that are broadly protective against CoVs
that infect humans, one may want to test whether the vaccine
composition binds to antibodies that bind to a broad CoV species.
When the purpose of vaccine is to induce immune responses mainly
against SARS-CoV and/or SARS-CoV-2, one may want to test whether
the vaccine composition binds to antibodies that bind to SARS-CoV
and/or SARS-CoV-2.
[0644] For this purpose of vaccine effectiveness prediction, a kit
that comprises at least one antibody and an instruction on how to
use such an antibody is envisioned. The instruction may teach how
to perform a vaccine efficacy prediction study.
[0645] Such a kit may also be for use in diagnosing CoV infection,
detection of the presence of CoV in a subject or a specimen from a
subject, treating CoV infection, or preventing CoV infection.
Example 11: Anti-CoV-S Antibody Screening
[0646] Additional antibodies or fragments thereof may be screening
or discovered using the antibody sequences disclosed herein. For
example, one or more antibodies comprising at least one of the six
CDRs of any one of the antibodies disclosed herein may be first
prepared. Then such antibody(ies) may be tested whether to provide
one or more of the flowing features: (i) binds to the S protein of
a CoV; (ii) binds to the S1 subunit of CoV-S; (iii) binds to the
RBD of CoV-S; (iv) binds to the NTD of CoV-S; (v) binds to the
ACE2-binding motif of CoV-S; (vi) competes with ACE2 such as hACE2;
(vii) competes with the antibody CR3022; (viii) neutralizes one or
more of SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-229E, HCoV-HKU1,
HCoV-NL63, or HCoV-OK43 or variants thereof; (iv) neutralizes a
pseudovirus of one or more of SARS-CoV, SARS-CoV-2, MERS-CoV,
HCoV-229E, HCoV-HKU1, HCoV-NL63, or HCoV-OK43 or variants thereof;
or (x) prevents or treats CoV infection in vivo. For such testing,
the method to be used is not limited to the method disclosed in the
current application but also may be any other test methods and/or
techniques of the field may be used.
[0647] It is noted that not only neutralizing antibodies but also
non-neutralizing antibodies may be useful for preventing and/or
treating CoV infection. For example, such non-neutralizing
antibodies can still mediate antibody dependent cell-mediated
cytotoxicity ("ADCC") and/or complement-dependent cytotoxicity
("CDC"), for example if the antibody binds to a CoV on the cell
surface. Furthermore, such non-neutralizing antibody or antigen
binding fragment thereof may be used as part of an ADC or a CAR
construct, because it is not necessarily the antigen-binding domain
that provides the cytotoxicity of an ADC or a CAR; rather it may
involve the drug that is conjugated or a host cell mechanism.
Accordingly, the antigen-binding domain may only need to bind to
CoV and does not by itself necessarily have to possess
neutralization capabilities. Moreover, non-neutralizing antibodies
may also be used for detecting CoV in a sample or to be used in
predicting vaccine effectiveness as described in Example 10.
Example 12: Affinity Maturation of SARS-CoV-2 Neutralizing
Antibodies
[0648] ADI-57983, ADI-57978, and ADI-56868 were obtained by
affinity maturation of ADI-55689; ADI-55688; and ADI-56046,
respectively.
[0649] Affinity maturation of antibodies was performed by
introducing diversities into the heavy chain and light chain
variable regions as described below.
[0650] VH CDR1, VH CDR2 and VH CDR3 selection: Forward priming
oligos were ordered from IDT with variegation in the VH CDR1, VH
CDR2, and VH CDR3 for heavy chain diversification. FR1-FR4 oligos
containing homology to the CDRs above were ordered in the reverse
priming direction for the assembly and amplification of the entire
heavy chain variable regions via PCR. The heavy chain variable
regions (FR1-FR4) were transformed into yeast containing the light
chain plasmid of the parent. With the library diversity of
1.times.10.sup.7, selections were performed with two rounds of
FACS, sorting for the highest affinity biotinylated SARS-CoV-2
spike protein binders using antigen titration.
[0651] VL CDR1, VL CDR2 and VL CDR3 selection: VL CDR1, VL CDR2,
and VL CDR3 diversification was obtained by ordering forward
priming oligos with variegation in each CDR. FR1-FR4 oligos
containing homology to the CDRs above were ordered in the reverse
priming direction for the assembly and amplification of the entire
light chain variable regions via PCR. The light chain variable
regions (FR1-FR4) were transformed into yeast containing the heavy
chain plasmid of the parent. With the library diversity of
1.times.10.sup.6, selections were performed with two rounds of
FACS. Affinity pressure was applied by titrating the biotinylated
SARS-CoV-2 spike protein.
[0652] Combined heavy chain and light chain selection (scheme shown
in FIG. 30C): The variable regions (FR1-FR4) from the highest
affinity IgGs of VH CDR1, VH CDR2, and VH CDR3 maturation cycle
were combined with the highest affinity IgGs of the VL CDR1, VL
CDR2, and VL CDR3 maturation cycle and transformed into yeast.
Selections were performed with two rounds of FACS, enriching for
the highest affinity biotinylated SARS-CoV-2 S1 protein binders. To
select for antibodies with better affinity than the input heavy
chain or light chain IgGs via FACs, the final sort population was
compared to the final rounds of the VH CDR1, VH CDR2, and VH
CDR3/VL CDR1, VL CDR2, and VL CDR3 maturation output. Affinity
matured progenies were assayed for the affinity for SARS-CoV-2 S
protein and for the SARS-CoV and SARS-CoV-2 neutralization ability
as described in Examples 13 and 14, and based on the results,
ADI-57983, ADI-57978, and ADI-56868, affinity-matured progenies of
ADI-55689, ADI-55688, and ADI-56046, respectively, were selected as
the best progenies.
[0653] FIG. 30D further shows that binding to the S1 subunit of
SARS-CoV-2-S is much higher in the affinity maturation progeny
library relative to the respective parent antibodies, ADI-55689,
ADI-55688, or ADI-56046.
[0654] The sequences of the VH and VL and CDRs and FRs of the VH
and VL of selected antibodies are provided in FIGS. 36A and 36B.
Sequence alterations relative to the germline or germline-encoded
sequences are caused by somatic mutation and alterations caused by
degenerate primers, are also referred to as "primer mutation"
herein.
Example 13: Kinetics of Binding Measurements (SARS-CoV-2-S) with
the Parent Antibodies and Affinity-Matured Progenies
[0655] The parent antibodies (i.e., before affinity maturation),
ADI-55689, ADI-55688, and ADI-56046, and Cycle 1 and Cycle 2
progenies generated via affinity maturation, including ADI-57983,
ADI-57978, and ADI-56868, obtained in in Example 12, were expressed
as a Fab using the method described in Example 1, and the apparent
binding affinities (K.sub.D.sup.Apps) of each Fab to
prefusion-stabilized SARS-CoV-2 S protein were determined by BLI (4
hour incubation) as described in Example 2. The results are
provided in FIG. 30A, which shows marked improvement (approximately
25- to 630-fold improvements) in the affinity to SARS-CoV-2-S of
Cycle 1 progenies over the respective parents and in Cycle 2
progenies over the respective Cycle 1 progenies.
Example 14: Micro-Titer Neutralization Assays with the Parent
Antibodies and Affinity-Matured Progenies
14-1: Authentic SARS-CoV and SARS-CoV-2 Neutralization Assay
[0656] Neutralization of authentic SARS-CoV and SARS-CoV-2 by the
parent antibodies (i.e., before affinity maturation), ADI-55689,
ADI-55688, and ADI-56046, and affinity matured progenies, including
ADI-57983, ADI-57978, and ADI-56868, obtained in Example 13, was
measured using the method described in 6-1 of Example 6. The
results are provided in FIG. 30B, which shows marked improvement in
the ability to neutralize both SARS-CoV and SARS-CoV-2 by affinity
maturation.
[0657] Based on the results in Examples 13 and 14, it was found
that improvements in SARS-CoV-2 S protein affinity translate to
improvements in neutralization. The IC50 values of ADI-57983,
ADI-57978, and ADI-56868 are also provided in FIG. 31A.
14-2: SARS-CoV-MLV and SARS-CoV-2-MLV Pseudo Viral Particle
Neutralization Assay
[0658] Neutralization of SARS-CoV and SARS-CoV-2 MLV pseudo viral
particle by ADI-57983, ADI-57978, and ADI-56868, obtained in
Example 13, was measured using the method described in 6-3 of
Example 6.
[0659] The results are provided in FIG. 30B, which shows highly
similar results to those from the authentic virus neutralization
studies in 14-1. The IC50 values of ADI-57983, ADI-57978, and
ADI-56868 are also provided in FIG. 31A.
Example 15: Selection and Preparation of Antibodies that
Selectively Bind SARS-CoV-2-S Using Blood Samples from Convalescent
COVID-19 Patients
[0660] ADI-56443 and ADI-56479 were identified as potent
neutralizing antibodies with high affinity for the RBD and NTD,
respectively, of the spike (S) protein of SARS-CoV-2. Sample
[0661] Heparinized blood (50-100 cc) was obtained from two subjects
(2019-2020 SARS-CoV-2 outbreak survivor) one month after infection
(Donor names: "EMC10" and "EMC15"). The sample was processed to
obtain plasma and to isolate peripheral blood-derived B cells.
Isolated cells and plasma were stored frozen in aliquots at
-80.degree. C.
Antigens and Antibodies
[0662] Production of recombinant SARS-CoV-2 spike protein: To
express the prefusion S ectodomain of SARS-CoV-2, a gene encoding
residues 1-1208 of 2019-nCoV S (GenBank: MN908947) with proline
substitutions at residues 986 and 987, a "GSAS" substitution
("GSAS" disclosed as SEQ ID NO: 23196) at the furin cleavage site
(residues 682-685), a C-terminal T4 fibritin trimerization motif,
an HRV3C protease cleavage site, a TwinStrepTag and an
8.times.HisTag (SEQ ID NO: 23197) was synthesized and cloned into
the mammalian expression vector paH (DOI: 10.1126/science.abb2507).
Expression construct design was based on previously described
strategies for expression of related betacoronavirus S proteins
(DOI: 10.1073/pnas.1707304114 and DOI: 10.1038/s41598-018-34171-7)
These expression vector encoding SARS-CoV-2 S protein was used to
transiently transfect FreeStyle293F cells (Thermo 134 Fischer)
using polyethylenimine. Protein was purified from filtered cell
supernatants using either 135 StrepTactin resin (IBA) or Protein A
resin (Pierce) before being subjected to additional 136
purification by size-exclusion chromatography using either a
Superose 6 10/300 column (GE 137 Healthcare) or a Superdex 200
10/300 Increase column (GE Healthcare) in 2 mM Tris pH 8.0, 138 200
mM NaCl and 0.02% NaN3. The final protein preparations were stored
in phosphate-buffered saline pH 7.4 supplemented with an additional
150 mM NaCl. Small aliquots were stored at -70.degree. C. until
use.
Single B-Cell Sorting
[0663] For MBC sorting, B cells were purified using a MACS B cell
isolation kit (Miltenyi Biotec; cat #130-091-151) and subsequently
stained using anti-human CD19 (PE-Cy7), CD3 (PerCP-Cy5.5), CD8
(PerCP-Cy5.5), CD14 (PerCP-Cy5.5), CD16 (PerCP-Cy5.5), IgM (BV711),
IgD (BV421), IgA (AF488), IgG (BV605), CD27 (BV510), CD71 (APC-Cy7)
and dual-labeled (APC and PE) SARS-CoV-2 spike protein tetramer (25
nM). Tetramer was prepared fresh for each experiment, and B cells
that showed reactivity to the SARS-CoV-2 spike protein tetramer was
single cell sorted. Single cells were sorted using a BD FACS Aria
II (BD Biosciences) into 96-well PCR plates (BioRAD) containing 20
uL/well of lysis buffer [5 uL of 5.times. first strand cDNA buffer
(Invitrogen), 0.625 uL of NP-40 (New England Biolabs), 0.25 uL
RNaseOUT (Invitrogen), 1.25 uL dithiothreitol (Invitrogen), and
12.6 uL dH2O]. Plates were immediately stored at -80.degree. C.
Flow cytometry data were analyzed using FlowJo software.
Amplification and Cloning of Antibody Variable Genes
[0664] Antibody variable genes (IgH, IgK, and IgL) were amplified
by reverse transcription PCR and nested PCRs using cocktails of
IgG- and IgM-specific primers, as described previously (Tiller et
al, J Immunol 2008). The primers used in the second round of PCR
contained 40 base pairs of 5' and 3' homology to the digested
expression vectors, which allowed for cloning by homologous
recombination into S. cerevisiae. The lithium acetate method for
chemical transformation was used to clone the PCR products into S.
cerevisiae (Gietz and Schiestl, Nat Protoc 2007). 10 uL of
unpurified heavy chain and light chain PCR product and 200 ng of
the digested expression vectors were used per transformation
reaction. Following transformation, individual yeast colonies were
picked for sequencing and characterization. Cognate antibody heavy-
and light-chain pairs were rescued from individual B cells and
cloned. The sequences, the germline origins, and isotypes were
determined. The number of nucleic acid substitutions relative to
the germline sequences and the number of amino acid alterations
relative to the germline-encoded sequences were also analyzed. The
sequences of the VH and VL and CDRs and FRs of the VH and VL of two
identified antibodies, ADI-56443 and ADI-56479, are provided in
FIGS. 36A and 36B. Sequence alterations relative to the germline
encoded sequences are shown in red (caused by somatic mutation) and
orange (caused by degenerate primers, also referred to as "primer
mutation" herein). Germline origins and isotypes are provided in
FIGS. 31 and 35.
Expression and Purification of IgGs and Fab Fragments
[0665] IgGs were expressed in S. cerevisiae cultures grown in
24-well plates, as described previously (Bornholdt et al, Science
2016, PMID:26912366). After 6 days, the cultures were harvested by
centrifugation and IgGs were purified by protein A-affinity
chromatography. The bound antibodies were eluted with 200 mM acetic
acid/50 mM NaCl (pH 3.5) into 1/8th volume 2 M Hepes (pH 8.0), and
buffer-exchanged into PBS (pH 7.0).
[0666] CR3022 was cloned into S. cerevisiae using recombinational
cloning. The variable region sequences of CR3022 were synthesized
as gBlock fragments (IDT) with homologous overhangs which were
cloned and expressed as described above.
Fab fragments were generated by digesting the IgGs with papain for
2 h at 30.degree. C. The digestion was terminated by the addition
of iodoacetamide, and the Fab and Fc mixtures were passed over
Protein A agarose to remove Fc fragments and undigested IgG. The
flowthrough of the Protein A resin was then passed over
CaptureSelect.TM. IgG-CH1 affinity resin (ThermoFischer Scientific)
and eluted with 200 mM acetic acid/50 mM NaCl pH 3.5 into 1/8th
volume 2M Hepes pH 8.0. Fab fragments then were buffer-exchanged
into PBS pH 7.0.
Example 16: Kinetics of Binding Measurements (S Protein of
SARS-CoV-2 and Seasonal CoV) with the Antibodies Isolated from
Covalescent COVID-19 Patients
Bio-Layer Interferometry Kinetic Measurements (BLI):
[0667] The apparent binding affinities (K.sub.D.sup.Apps) of
ADI-56443 and ADI-56479, obtained in in Example 15, to
prefusion-stabilized S protein of SARS-CoV-2 or HKU1 were
determined by BLI (4 hour incubation) as described in Example 2.
The results are provided in FIGS. 32B and 32G, which shows that
both ADI-56443 and ADI-56479 bind to SARS-CoV-2-S but neither
ADI-56443 nor ADI-56479 binds to HKU1-S.
FRNT Assay:
[0668] ADI-56443 and ADI-56479, obtained in in Example 15, were
expressed as human IgG as described in Example 15, and the apparent
binding affinities (K.sub.D.sup.ApPs) for SARS-CoV-S and
SARS-CoV-2-S protein were determined.
[0669] For SARS CoV or SARS-CoV-2 spike protein binding ELISAs,
96-well plates (Corning; Cat #3690) were coated with 5 .mu.g/ml of
SARS CoV or SARS-CoV-2 spike protein diluted in PBS and incubated
overnight at 4.degree. C. Wells were washed and then blocked with
5% non-fat dried milk (NFDM) in PBS for 1 hour at 37.degree. C.
Wells were washed 3 times with PBS and serial dilutions of human
plasma in 5% NFDM-PBS were added and incubated for 1 hour at
37.degree. C. Plates were then washed 3 times with PBS and
secondary cross-adsorbed anti-human IgG-HRP (Thermo Fisher
Scientific; cat #31413) or anti-human-IgM (Sigma Aldrich; cat
#AP114P) detection antibodies were added at 1:8000 dilution in 5%
NFDM-PBS for 1 hour at 37.degree. C. After washing 3 times with PBS
detection reagent was added per manufacturer recommendations
(Thermo Scientific; Cat #34029) and absorbance was measures at 450
nM wavelength using a Spectramax microplate Reader (Molecular
Devices).
[0670] The results showed that both ADI-56443 and ADI-56479 bind to
SARS-CoV-2-S (data not shown).
Example 17: Micro-Titer Neutralization Assays with ADI-56443 and
ADI-56479
17-1: Authentic SARS-CoV-2 Neutralization Assay
[0671] Neutralization of authentic SARS-CoV-2 by ADI-56443 and
ADI-56479, obtained in Example 15, was measured using the method
described in 6-1 of Example 6. The results are provided in FIGS.
31A and 33. Both ADI-56443 and ADI-56479 efficiently neutralized
authentic SARS-CoV-2.
17-2: rVSV-SARS-CoV-2 Neutralization Assay
[0672] Neutralization of SARS-CoV-2 rVSV pseudo viral particle by
ADI-56443 and ADI-56479, obtained in Example 15, was measured using
the method described in 6-2 of Example 6.
[0673] The results are provided in FIGS. 31A and 33. Both ADI-56443
and ADI-56479 efficiently neutralized SARS-CoV-2 rVSV
pseudoviruses.
Example 18: Developability Profile Analysis
[0674] For each of ADI-57983, ADI-57978, and ADI-56868, obtained in
Example 12, and ADI-56443 and ADI-56479, obtained in Example 15,
the acceptability for multiple biophysical metrics of
"developability" were assessed using a panel of assays: PSR, HIC,
and Tm.
[0675] Polyspecificity (also referred to as polyreactivity) is a
highly undesirable property that has been linked to poor antibody
pharmacokinetics (Wu et al., J Mol Biol 368:652-665, 2007; Hotzel
et al., 2012, MAbs 4(6):753-760) and, thus, potentially to poor
developability. Antibodies can be detected as possessing decreased
or increased developability by virtue of their level of interaction
with polyspecificity reagent (PSR). See WO2014/179363. Antibodies
displaying increased interaction with PSR are referred to as
"polyspecific" polypeptides, with poor(er) developability. SARS2
antibodies selected or identified as possessing enhanced
developability based on low polyspecificity score are considered
"developable".
[0676] A poly specificity of each antibody was measured as
described previously (L. Shehata et al., Affinity Maturation
Enhances Antibody Specificity but Compromises Conformational
Stability. Cell reports 28, 3300-3308 e3304 (2019)). Briefly,
soluble membrane protein (SMP) and soluble cytosolic protein (SCP)
fractions obtained from Chinese hamster ovary (CHO) cells were
biotinylated using NHS-LC-Biotin (Thermo Fisher Scientific Cat
#21336). IgGs presented on the surface of yeast were incubated with
1:10 diluted biotinylated CHO cell preparations on ice for 20
minutes. Cells were then washed twice with ice-cold PBS containing
0.1% BSA (PBSF) and incubated in 50 .mu.L of a secondary labelling
mix containing ExtrAvidin-R-PE (Sigma-Aldrich), anti-human LC-FITC
(Southern Biotech) and propidium iodide) for 15 minutes. The cells
were washed twice with PBSF and resuspended in PBSF to be run on a
FACSCanto II (BD Biosciences). The mean fluorescence intensity of
binding was normalized using control antibodies that display low,
medium or high polyspecificity to assess the non-specific binding.
The results are summarized in FIG. 31B.
[0677] The polyspecificity score can be useful, as one or one of
many metrics, in ranking the antibodies based on developability.
For example, the antibodies can be ranked as clean (below 0.11),
low (below 0.33), medium (below 0.66), and high polyspecificity
(above 0.66). The antibodies were categorized as A when the Score
is below 0.10. and as B when the Score is 0.10 or higher. All
tested antibodies were "A".
[0678] Hydrophobic interaction chromatography (HIC) was performed
to assess hydrophobic interaction of the lead antibodies. The
methodology for this assay was described previously (see Estep P,
et al. (2015) An alternative assay to hydrophobic interaction
chromatography for high-throughput characterization of monoclonal
antibodies. MAbs 7(3):553-561). In brief, 5 .mu.g IgG samples (1
mg/mL) were spiked in with a mobile phase A solution (1.8 M
ammonium sulfate and 0.1 M sodium phosphate at pH 6.5) to achieve a
final ammonium sulfate concentration of about 1 M before analysis.
A Sepax Proteomix HIC butyl-NP5 column was used with a liner
gradient of mobile phase A and mobile phase B solution (0.1 M
sodium phosphate, pH 6.5) over 20 min at a flow rate of 1 mL/min
with UV absorbance monitoring at 280 nm. Results are summarized in
FIG. 31B.
[0679] Lastly, Tm was measured using DSF. The Tm was determined
using a CFX96 Real-Time System from Bio-Rad, based on the protocol
described earlier (32). Briefly, 20 .mu.L of 1 mg/mL sample was
mixed with 10 .mu.L of 20.times.SYPRO orange. The plate was scanned
from 40.degree. C. to 95.degree. C. at a rate of 0.5.degree. C./2
min. The Fab Tm was assigned using the first derivative of the raw
data from the Bio-Rad analysis software. Results are summarized in
FIG. 31B. FIG. 31B further provides pI (isoelectric point)
values.
[0680] Collectively, ADI-57983, ADI-57978, ADI-56868, ADI-56443,
and ADI-56479 have favorable developability profiles.
Example 19: Epitope Mapping
[0681] Epitope mapping was performed for ADI-57983, ADI-57978, and
ADI-56868, obtained in Example 12, and ADI-56443 and ADI-56479,
obtained in Example 15, using different subunit and domain
constructs of SARS-CoV-2-S using ForteBio-based competition
assays.
[0682] The domain/subunit used for the binding studies were the S1
subunit, S2 subunit, the RBD, and NTD and for the binding to RBD
and NTD, monovalent binding and bivalent ("AVID") binding were both
measured.
[0683] For the competition studies, the competition of each
antibody with recombinant human ACE2 ("hACE2") and with a
commercial antibody CR3022, which targets a conserved epitope
outside of the receptor binding site, was tested (M. Yuan et al., A
highly conserved cryptic epitope in the receptor-binding domains of
SARS-CoV-2 and SARS-CoV. Science, (2020)).
[0684] Results are summarized in FIGS. 31A and 32A. ADI-57983,
ADI-57978, ADI-56868, and ADI-56443 recognized epitopes within the
RBD and compete with hACE2, whereas ADI-56479 recognized an epitope
in the NTD. Binding data for ADI-56443 and ADI-56479 are also
provided in FIGS. 32B-32G, and the competition results for
ADI-56443 is provided in FIG. 32H.
Example 20: Cross Competition
[0685] Cross-competition of antibodies for binding to recombinant
SARS-CoV-2 RBD was evaluated using a ForteBio Octet HTX instrument
(Molecular Devices). All binding steps were performed at 25.degree.
C. and at an orbital shaking speed of 1000 rpm. All reagents were
formulated in PBSF buffer (PBS with 0.1% w/v BSA). The IgGs (100
nM) were captured to anti-human IgG capture (AHC) biosensors
(Molecular Devices) to a sensor response of 1.0 nm-1.4 nm, the
remaining unoccupied binding sites on the biosensor were blocked
with an inert IgG (0.5 mg/mL), and then allowed to equilibrate in
PBSF for a minimum of 30 min. To assess any cross interactions
between proteins on the sensor surface and the secondary molecules,
the loaded and blocked sensors were exposed (90 s) to competitor
IgG (300 nM) prior to the binning analysis. After a short baseline
step (60 s) in PBSF, the IgG-loaded biosensor tips were exposed
(180 s) to the SARS-CoV-2 RBD-SD1 antigen (100 nM) and then exposed
(180 s) to competitor IgG (300 nM). The data was y-axis normalized,
and interstep corrected using the FortdBio Data Analysis Software
version 11.0. Additional binding by the secondary molecule
indicates an unoccupied epitope (non-competitor), while no binding
indicates epitope blocking (competitor). Antibodies with a binding
response value of <0.1 were considered to be non-binders.
[0686] Notably, ADI-56443 does not compete with ADI-55689,
ADI-55688, and ADI-56046 (FIGS. 321 and 32J) and, likewise, are not
expected to compete with ADI-57983, ADI-57978, ADI-56868.
Example 21: Primer Mutation Fix
[0687] As described above in Examples 12 and 15, ADI-57983,
ADI-57978, and ADI-56868, obtained in Example 12, and ADI-56443 and
ADI-56479, obtained in Example 15, contained at least one "primer
mutation" caused by the use of degenerate primers. The "primer
mutation(s)" in the variable region sequences of ADI-57983,
ADI-57978, ADI-56868, ADI-56443, and ADI-56479 were fixed back to
the germline-encoded amino acid(s), and the resulting antibodies
were named ADI-58120, ADI-58124, ADI-58126, ADI-58128, and
ADI-58130, respectively. Amino acid changes are also explained in
FIG. 34. The VH and VL sequences of ADI-58120, ADI-58124,
ADI-58126, ADI-58128, and ADI-58130 are provided in FIGS. 36A and
36B, and the number of differences in the amino acids and
nucleotides relative to the germline-coded or germline sequences is
summarized in FIG. 35.
[0688] Additionally, a variant of ADI-58130, which differs from
AD-58130 by only one amino acid in the variable region sequence,
was obtained and named ADI-58130-LCN30cQ. The variant has "Q"
instead of "N" in the light chain CDR. The variable region
sequences of ADI-58130-LCN30cQ are also provided in FIGS. 36A and
36B).
[0689] The variable region sequences of any of these six antibodies
(ADT-58120, ADI-58124, ADI-58126, AD-58128, and ADI-58130, and
ADI-58130-LCN30cQ) or any other antibodies described herein may be
used with a wild type or variant Fc region. Examples of Fc variants
including wild-type Fc are provided in Table 1. Heavy and light
chain sequences of exemplary antibodies using the variable region
sequences of ADI-58120, ADT-58124, ADI-58126, ADI-58128, or
AD-58130 and one of the Fc sequences provided in Table 1 are
provided in Table 2, and separate ADI IDs are assigned for
respective antibodies (see FIGS. 36A and 36B for SEQ ID NOs).
TABLE-US-00002 TABLE 2 Exemplary Antibodies With Different Variable
Region Sequences And Fc Sequences. ADI ID Variable Region Same As
Fc Variant Name ADI-58120 ADI-58120 WT ADI-58121 ADI-58120 YTE
ADI-58122 ADI-58120 LA ADI-58123 ADI-58120 LS Not assigned
ADI-58120 LA-RE ADI-58124 ADI-58124 WT ADI-58125 ADI-58124 LA
ADI-58126 ADI-58126 WT ADI-58127 ADI-58126 LA ADI-58128 ADI-58128
WT ADI-58130 ADI-58130 WT ADI-58129 ADI-58128 LA ADI-58131
ADI-58130 LA ADI-58130_LCN30cQ ADI-58130_LCN30cQ WT ADI-59988
ADI-58130_LCN30cQ LA ADI-58128 ADI-58128 WT
Example 22: Affinity of Post-Affinity Maturation Antibodies.
PGP-23.T1
[0690] The affinity of ADI-58120, ADI-58124, and ADI-58126 to
SARS-CoV-2 RBD-SD1 (SD1 is the subdomain 1) and the respective
parent antibodies before affinity maturation was measured using SPR
as described below. As shown in FIG. 37A, the post-affinity
maturation antibodies showed 25 to 630-fold improvements in binding
relative to their respective parental antibodies.
[0691] The affinity of ADI-58124 and its parental antibodies
(before and after affinity maturation) expressed as Fab to
SARS-CoV-2 S protein, measured by BLI, is shown in FIG. 37B.
[0692] The affinity of ADI-58125 expressed as IgG or Fab to
SARS-CoV-2 S protein, SARS-CoV-2 S protein RBD, SARS-CoV S protein,
SARS-CoV S protein RBD, and WIV-1 S protein RBD was also measured
via SPR as described below. Binding kinetics results are summarized
in FIG. 37C.
[0693] A brief description of methods used is provided below.
[0694] Expression and Purification of IgGs and Fab Fragments:
[0695] Monoclonal antibodies were produced as full-length IgG1s in
S. cerevisiae cultures, as described in Wec A. et al., Science.
2020 Jun. 15; eabc7424. Briefly, yeast cultures were incubated in
24-well plates placed in Infors Multitron shaking incubators at
30.degree. C., 650 rpm and 80% relative humidity. After 6 days, the
supernatants containing the IgGs were harvested by centrifugation
and purified by protein A-affinity chromatography. The bound IgGs
were eluted with 200 mM acetic acid/50 mM NaCl (pH 3.5) into 1/8th
volume 2 M Hepes (pH 8.0) and buffer-exchanged into PBS (pH
7.0).
[0696] Fab fragments for structural studies were also generated as
described in Wec A. et al., Science. 2020 Jun. 15; eabc7424.
Briefly, IgGs were digested with papain for 2 hours at 30.degree.
C. followed by addition of iodoacetamide to terminate the
digestion. To remove the Fc fragments and any undigested IgG
fractions, the mixtures were passed over Protein A agarose. The
flow-through of the Protein A resin was then passed over
CaptureSelect.TM. IgG-CH1 affinity resin (ThermoFisher Scientific)
and the captured Fabs were eluted with 200 mM acetic acid/50 mM
NaCl (pH 3.5) into 1/8th volume 2 M Hepes (pH 8.0) followed by
buffer exchange into PBS (pH 7.0).
[0697] Surface Plasmon Resonance (SPR) Fab Kinetic Binding
Measurements:
[0698] SEC-purified SARS-CoV-2 RBD-SD1 was immobilized to a NiNTA
sensor chip in a Biacore X100 (GE Life Sciences) to a response
level of .about.500 RUs. Fabs were then injected at increasing
concentrations, ranging from 18.75-300 nM (ADI-55688), 1.56-25 nM
(ADI-56046), 6.25-100 nM (ADI-55689), or 1.25-20 nM (ADI-58120,
ADI-58124, ADI-58126). The sensor chip was doubly regenerated
between cycles using 0.35 M EDTA and 0.1 M NaOH. The resulting data
were double reference subtracted and fit to a 1:1 binding model
using Biacore Evaluation Software.
[0699] In the next several examples, ADI-58120, ADI-58124,
ADI-58126, ADI-58128, ADI-58130, and/or ADI-58130-LCN30cQ, and/or
their variants comprising a non-wild-type Fc region were compared
with antibodies in the clinic that are specific to SARS-CoV-2.
Table 3 provides the sequences of VH, VL, HC, and LC, respectively,
of anti-SARS-CoV-2 antibodies currently under clinical trials.
TABLE-US-00003 TABLE 3 Sequences for Clinical Antibodies Antibody
Fc LC name VH VL HC variant LC Class S309 SEQ ID SEQ ID SEQ ID LS
SEQ ID kappa NO: 22 NO: 32 NO: 21 NO: 31 REGN10987 SEQ ID SEQ ID
SEQ ID WT-DEL SEQ ID lambda NO: 42 NO: 52 NO: 41 NO: 51 REGN10933
SEQ ID SEQ ID SEQ ID WT-DEL SEQ ID kappa NO: 62 NO: 62 NO: 61 NO:
61 JS016 SEQ ID SEQ ID SEQ ID LALA- SEQ ID kappa NO: 82 NO: 92 NO:
81 DEL NO: 91
Example 23: Antibody Developability of Post-Affinity Maturation
Antibodies
[0700] Because in vitro engineering can lead to polyspecificity
with potential risks of off-target binding and accelerated
clearance in vivo (S. A. Sievers, et al., Curr Opin HIV AIDS 10,
151-159 (2015)), we assessed the polyspecificity of ADI-58120,
ADI-58124, ADI-58126, ADI-58128, and ADI-581230, and Fc variants
thereof using a previously described assay that has been shown to
be predictive of serum-half life in humans (L. Shehata et al., Cell
Rep 28, 3300-3308 e3304 (2019)). All three antibodies lacked
polyreactivity in this assay, indicating a low risk for poor
pharmacokinetic behavior (FIG. 38A). The three antibodies also
showed low hydrophobicity, a low propensity for self-interaction,
and thermal stabilities within the range observed for clinically
approved antibodies (FIGS. 38A-38D), suggesting that the process of
in vitro engineering did not negatively impact biophysical
properties that are often linked to down-stream behaviors such as
ease of manufacturing, ability to formulate to high concentrations,
and long-term stability.
[0701] Brief descriptions of the methods used are provided
below:
[0702] Polyreactivity Assay:
[0703] Polyspecificity reagent binding of antibodies was performed
as described previously (8). Briefly, soluble membrane protein
(SMP) and soluble cytosolic protein (SCP) fractions were extracted
from Chinese hamster ovary (CHO) cells and biotinylated using
NHS-LC-Biotin (Thermo Fisher Scientific) reagent. Yeast-presented
IgGs were incubated with 1:10 diluted stock of biotinylated SMP and
SCP for 20 minutes on ice, followed by two washes with PBSF, and
stained with 50 .mu.l of the secondary labelling mix containing
ExtrAvidin-R-PE (Sigma-Aldrich), anti-human LC-FITC (Southern
Biotech), and propidium iodide (Invitrogen) for 15 minutes on ice.
Cells were subsequently washed out of secondary reagents with PBSF
and resuspended in PBSF for flow cytometric analysis on a BD FACS
Canto II (BD Biosciences).
[0704] Affinity-Capture Self-Interaction Nanoparticle Spectroscopy
(AC-SINS):
[0705] To measure the propensity for antibodies to self-associate,
AC-SINS was performed based on a previously described protocol (Liu
Y et al., MAbs. March-April 2014; 6(2):483-92). Briefly, polyclonal
goat anti-human IgG Fc antibodies (capture; Jackson ImmunoResearch
Laboratories) and polyclonal goat non-specific antibodies
(non-capture; Jackson ImmunoResearch Laboratories) were buffer
exchanged into 20 mM sodium acetate (pH 4.3) and concentrated to
0.4 mg/ml. A 4:1 volume ratio of capture:non-capture was prepared
and further incubated at a 1:9 volume ratio with 20 nm gold
nanoparticles (AuNP; Ted Pella Inc.) for 1 hour at room
temperature. Thiolated PEG (Sigma-Aldrich) was then used to block
empty sites on the AuNP and filtered via a 0.22 m PVDF membrane
(Millipore). Coated particles were subsequently added to the test
antibody solution and incubated for 2 hours at room temperature
before measuring absorbance from 510 to 570 nm on a plate reader.
Data points were fit with a second-order polynomial in Excel to
obtain wavelengths at maximum absorbance. Values are reported as
the difference between plasmon wavelengths of the sample and
background (.DELTA..lamda.max).
[0706] Fab Melting Temperature:
[0707] Apparent melting temperatures (TmApp) of Fab fragments were
obtained as previously described (He F. et al., J Pharm Sci. 2011
December; 100(12):5126-41). Briefly, 20 .mu.l of test antibody
solution at 1 mg/ml was mixed with 10 .mu.l of 20.times.SYPRO
orange. The plate was scanned with a CFX96 Real-Time System
(BioRad) from 40.degree. C. to 95.degree. C. at a rate of
0.25.degree. C. per minute. TmApp was calculated from the primary
derivative of the raw data via the BioRad analysis software.
[0708] Hydrophobic Interaction Chromatography (HIC):
[0709] Antibody hydrophobicity was evaluated using HIC as
previously described (Estep P. et al., MAbs. 2015; 7(3):553-61).
Test antibody samples were diluted in phase A solution (1.8 M
ammonium sulfate and 0.1 M pH 6.5 sodium phosphate) to a final
concentration of 1.0 M ammonium sulfate. A linear gradient from
phase A solution to phase B solution (0.1 M pH 6.5 sodium
phosphate) was run for 20 minutes at a flow rate of 1.0 ml/min
using the Sepax Proteomix HIC butyl-NP5 column. Peak retention
times were obtained from monitoring UV absorbance at 280 nm.
Example 24: Neutralization Potential of Post-Affinity Maturation
Antibodies
[0710] To determine whether the improvements in SARS-CoV-2 S
binding affinity translated into enhanced neutralization potency,
we selected between 9 and 14 affinity-matured progeny from each
lineage (including ADI-58120, ADI-58124, and ADI-58126) and
evaluated them for SARS-CoV-2 neutralizing activity in a murine
leukemia virus (MLV) pseudovirus assay (T. Giroglou, et al., J
Virol 78, 9007-9015 (2004)). The neutralizing activities of several
clinical neutralizing antibodies (nAbs) were also measured (S309,
REGN10933, REGN10987, and CB6/JS016) as benchmarks (D. Pinto et
al., Nature 583, 290-295 (2020).; R. Shi et al., Nature 584,
120-124 (2020).; J. Hansen et al., Science 369, 1010-1014 (2020)).
All of the affinity matured antibodies showed improved neutralizing
activity relative to their parental clones, and the most potent
neutralizers from each lineage (ADI-58120, ADI-58124, and
ADI-58126) displayed neutralization IC50s that were comparable to
or lower than those observed for the clinical SARS-CoV-2 nAb
controls (FIG. 39A).
Example 25: Neutralization Breadth of Post-Affinity Maturation
Antibodies
[0711] To determine whether the process of SARS-CoV-2 affinity
engineering impacted neutralization breadth, ADI-58120, ADI-58124,
and ADI-58126, as well as their respective parental antibodies,
were evaluated for neutralizing activity against a panel of
representative authentic clade I sarbecoviruses (SARS-CoV, SHC014,
SARS-CoV-2, and WIV-1). Consistent with the MLV-SARS-CoV-2 assay
results, ADI-58124 displayed highly potent neutralizing activity
against authentic SARS-CoV-2, with an IC50 comparable to or lower
than that observed for the clinical SARS-CoV-2 nAbs (FIGS.
39B-39D). Furthermore, in contrast to the clinical nAbs, ADI-58124
also displayed remarkable neutralization potency against SARS-CoV
and the two SARS-related bat viruses, with IC50s between 4-8 ng/ml
(FIGS. 39B-39D). Notably, ADI-58126 and the clinical nAb S309 also
cross-neutralized all four sarbecoviruses, but with markedly lower
potency than ADI-58124. Finally, ADI-58120 potently neutralized
SARS-CoV-2, SARS-CoV, and WIV1, but it lacked activity against
SHC014.
[0712] Based on its potent cross-neutralization and favorable
biophysical properties, ADI-58124 and ADI-58125 were selected for
further assessing its neutralizing activity in two alternative
authentic SARS-CoV-2 neutralization assays, which confirmed its
high potency (IC50.about.1 ng/ml) (FIGS. 39B-39E, FIG. 39N and FIG.
39O). Interestingly, ADI-58124, ADI-58125, CB6/JS016, REGN10987 and
REGN10933 reached 100% neutralization on both Vero and HeLa-ACE2
target cells in this assay, whereas S309 showed complete
neutralization on Vero target cells but plateaued at approximately
40% neutralization on HeLa-ACE2 target cells (FIGS. 39E and 39O).
S309 also failed to neutralize MLV-SARS-CoV-2 on HeLa-ACE2 target
cells (FIG. 39A). The reason for this is unclear but may relate to
glycan heterogeneity within the S309 epitope (D. Pinto et al.,
Nature 583, 290-295 (2020)) coupled with differences in receptor
expression or protease cleavage efficiency between the two types of
target cells (T. F. Rogers et al., Science 21, 956-963 (2020)).
Because SARS-CoV-2 D614G has emerged as the dominant pandemic form
(B. Korber et al., Cell 182, 812-827 e819 (2020)), ADI-58124 was
also evaluated for neutralizing activity against this variant in
the MLV pseudovirus assay. As expected, based on the location of
the D614G substitution outside of the RBD, ADI-58124 neutralized
the D614G variant with equivalent potency as wild-type (WT)
SARS-CoV-2 (FIG. 39F).
[0713] Neutralization of authentic and pseudo coronaviruses by
ADI-58120, ADI-58124, and ADI-58126, and/or their variants having a
non-wild type Fc are provided in FIGS. 39G-M.
[0714] Brief description of methods used is provided below.
[0715] HeLa-hACE2 Stable Cell Line:
[0716] Stable human ACE2 (hACE2)-expressing HeLa cells for
authentic SARS-CoV-2 neutralization assays were generated as
previously described (Wec A. et al., Science. 2020 Jun. 15;
eabc7424). Briefly, hACE2 (NM_001371415) was cloned into pBOB and
co-transfected with lentiviral vectors pMDL (Addgene #12251), pREV
(Addgene #12253), and pVSV-G (Addgene #8454) into HEK293T cells
using Lipofectamine 2000 (Thermo Fisher Scientific) according to
the manufacturer's protocol. Culture media was exchanged 16 hours
post-transfection, and supernatant containing hACE2 lentiviruses
was harvested 32 hours post-transfection. Pre-seeded HeLa cells
were transduced using harvested supernatant with 10 .mu.g/ml
polybrene (Sigma). At 12 hours post-transduction, cell surface
expression was confirmed by flow cytometry using SARS-CoV-2 S-based
probes.
[0717] Generation of Authentic SARS-CoV Virus and Neutralization
Assay:
[0718] To generate authentic SARS-CoV virus, Vero African grivet
monkey kidney cells (Vero E6, ATCC-CRL1586) were grown in
Dulbecco's Modified Eagle Medium (DMEM high glucose; Gibco, Cat.
#11995065), 2% heat-inactivated fetal bovine serum (FBS, Atlanta
Biologicals), 0.05% Trypsin-EDTA solution (Gibco), 1% PS (Gibco),
and 1% GlutaMAX (Gibco). Vero E6 cells were infected with
SARS-CoV/Urbani (MOI=0.01) and incubated at the following
conditions: 37.degree. C., 5% CO.sub.2 and 80% relative humidity
(RH). At 50 hours post-infection, cells were frozen at -80.degree.
C. for 1 hour and then thawed at room temperature. The supernatant
was collected and clarified by centrifugation at 2500.times.g for
10 minutes before aliquoting for storage at -80.degree. C.
[0719] Virus neutralization was assessed as previously described
(Wec A. et al., Science. 2020 Jun. 15; eabc7424. doi:
10.1126/science.abc7424). Briefly, SARS-CoV/Urbani (MOI=0.2) was
added to serial dilutions of antibodies and incubated for 1 hour at
room temperature. The antibody-virus mixture was added to
monolayers of Vero E6 cells in a 96-well plate and incubated for 1
hour at 37.degree. C., 5% CO.sub.2 and 80% RH. Next, media was
exchanged by washing cells once with 1.times.PBS and adding fresh
cell culture media. At 24-hour post-infection, cells were washed
out of media with 1.times.PBS to then be treated with formalin
fixing solution, permeabilized with 0.2% Triton-X for 10 minutes at
room temperature, and finally treated with blocking solution. Fixed
and permeabilized cells were first stained with a primary antibody
recognizing SARS-CoV nucleocapsid protein (Sino Biological),
followed by secondary antibody staining with AlexaFluor
488-conjugated goat anti-rabbit antibody. Infected cells were
enumerated by an Operetta high content imaging instrument and data
was analyzed using the Harmony software (Perkin Elmer).
[0720] Generation of MLV Pseudovirus Displaying SARS-CoV-2 WT and
D614G S and Neutralization Assay:
[0721] To generate the MLV pseudoviruses, pCDNA3.3 plasmids (Thermo
Fisher) encoding codon-optimized sequences of the SARS-CoV-2
wildtype (WT; NC_045512) and the D614G variant spike genes (IDT),
both with a 28 amino acid deletion in the C-terminal; a luciferase
reporter gene plasmid (Addgene #18760) modified with a CMV promoter
to replace the IRES; and the MLV Gag-Pol plasmid (Addgene #14887)
were purified using the Endo-Free Plasmid Maxi Kit (Qiagen). To
generate single-round infection competent pseudoviruses, HEK293T
cells were co-transfected with 2 gg of MLV Gag-Pol, 2 .mu.g of MLV
luciferase, and 0.5 .mu.g of either SARS-CoV-2 WT S or SARS-CoV-2
D614G S in 6-well plates using Lipofectamine 2000 (Thermo Fisher
Scientific) according to the manufacturer's directions. Cell
culture media was exchanged 16 hours post-transfection. At 48 hours
post-transfection, the supernatant containing SARS-CoV-2
S-pseudotyped viral particles was harvested, aliquoted, and frozen
at -80.degree. C.
[0722] Antibody neutralization of MLV-based SARS-CoV-2 WT and D614G
pseudoviruses was assessed via monitoring infection of HeLa-hACE2
cells by these pseudoviruses using a luminescence assay as
previously described (Sarzotti-Kelsoe M. et al., J Immunol Methods.
2014 July; 409:131-46.) with minor modifications. SARS-CoV-2 WT or
D614G pseudotyped MLV vector was mixed with serially diluted
antibodies, incubated for 1 hour at 37.degree. C., and followed by
addition of 10,000 HeLa-hACE2 cells. Infection was allowed to occur
for 42 to 48 hours at 37.degree. C. HeLa-hACE2 cells were
subsequently lysed using 1.times. luciferase lysis buffer (25 mM
Gly-Gly pH 7.8, 15 mM MgSO4, 4 mM EGTA, 1% Triton X-100).
Luciferase intensity was measured with a luminometer using
Bright-Glo luciferase substrate (Promega, PR-E2620) following
manufacturer's directions. Percentage of neutralization was
calculated as 100*(1-[Relative units of light (RUL) of
sample-Average RULs of background]/[Average RULs of virus-only
control-Average RULs of background]).
[0723] Generation of Nano-Luciferase (nLuc) Virus by CoV Reverse
Genetics:
[0724] Mouse-adapted SARS-CoV-1 (MA15), mouse adapted SARS-CoV-2
(MA2) and wildtype SARS-CoV-2 nano-luciferase (nLuc) viruses were
generated by CoV reverse genetics as described previously (Hou Y.
J. et al., Cell. 2020 Jul. 23; 182(2):429-446.e14.). WIV-1-nluc and
SHC014-nluc were generated by replacing ORF7 and 8 with nLuc. nLuc
viruses were subsequently used in luciferase-based antibody
neutralization assays on Vero E6 target cells as described
below.
[0725] Authentic SARS-CoV-2. WIV-1, and SHC014 Nano-Luciferase
Neutralization Assays:
[0726] Vero E6 cells were grown in DMEM high glucose media (Gibco
Cat. #11995065) supplemented with 10% fetal clone II (GE Cat.
#SH3006603HI)+1% non-essential amino acid and 1% Pen/Strep (growth
media) at 37.degree. C., 5% CO.sub.2. Vero E6 cells were seeded at
2.times.10.sup.4 cells/well in a black-wall, tissue culture
treated, 96-well plate (Corning, Cat #3603) 24 h before the assay.
A pre-determined amount of plaque-forming units (PFU) from a viral
titration curve was diluted in growth media. Antibodies were
diluted in growth media to obtain an 8-point, 3-fold dilution curve
with starting concentration at either 15, 7.5, 3.75 or 0.74
.mu.g/ml. SARS1-MA15-nLuc (75 Pfu/well), SARS2-nLuc (100 Pfu/well),
SARS2-MA2-nLuc (85 Pfu/well), SHCO14-nLuc (20 Pfu/well) and
WIV1-nLuc (250 Pfu/well) viruses were mixed with Abs at a 1:1 ratio
and incubated at 37.degree. C. for 1 h. Virus and Ab mix was added
to each well and incubated at 37.degree. C., 5% CO.sub.2 for 48 h
(SARS1-MA15, SARS2-MA2 and SARS2-nLuc) or 24 h (SHCO14-nLuc and
WIV1-nLuc). Luciferase activities were measured by the Nano-Glo
Luciferase Assay System (Promega Cat. #N1130) following the
manufacturer's protocol using a SpectraMax M3 luminometer
(Molecular Device). Percent inhibition was calculated by the
following equation: [1-(RLU with sample/RLU with mock
treatment)].times.100%. Fifty percent inhibition titer (IC.sub.50)
was calculated in GraphPad Prism 8.3.0 by fitting the data points
using a sigmoidal dose-response (variable slope) curve. All the
live virus experiments were performed under biosafety level 3
(BSL-3) conditions at negative pressure, by operators in Tyvek
suits wearing personal powered-air purifying respirators.
[0727] Generation of Authentic SARS-CoV-2 Virus and Neutralization
Assays:
[0728] Authentic SARS-CoV-2 virus was produced in Vero E6 cells as
described previously (Rogers T. F. et al., Science. 2020 Aug. 21;
369(6506):956-963). Briefly, Vero E6 cells were grown overnight in
complete DMEM (Corning, Cat #15-013-CV) supplemented with 10% FBS,
1.times.PenStrep (Corning, Cat #C20-002-CL), 2 mM (Corning, Cat
#25-005-CL) at 37.degree. C., 5% CO.sub.2. Cells were incubated
with two ml of SARS-CoV-2 strain USA-WA1/2020 (BEI Resources, Cat
#NR-52281) at multiplicity of infection of 0.5 for 30 min at
34.degree. C., 5% CO.sub.2, followed by direct addition of 30 ml of
complete DMEM. At 5 days post-infection, the supernatant was
centrifuged at 1000.times.g for 5 minutes, filtered using 0.22
.mu.M filters, and frozen at -80.degree. C.
[0729] Antibody neutralization against live, replicating, authentic
SARS-CoV-2 was assessed using 2 cell-based assays. Vero E6 cells
and a HeLa-hACE2 stable cell line were grown in complete DMEM
(Corning, Cat #15-013-CV) supplemented with 10% FBS,
1.times.PenStrep (Corning, Cat #C20-002-CL), 2 mM L-glutamine
(Corning, Cat #25-005-CL) at 37.degree. C., 5% CO.sub.2. Either
HeLa-hACE2 or Vero E6 target cells were seeded in a 96-well
half-well plate at approximately 8000 cells/well suspended in 50
.mu.l complete DMEM (Corning, Cat #15-013-CV) and grown overnight.
1000 plaque forming units (PFU)/well of SARS-CoV-2 was added to
titrating amounts of antibody and incubated for 30 minutes. The
virus-antibody mixture was subsequently incubated with either
HeLa-hACE2 or Vero E6 cells for 24 hours at 37.degree. C., 5%
CO.sub.2. Following incubation, the infection media was removed.
Cells were submerged in 4% formaldehyde for 1 hour, followed by
three cycles of washing with PBS, and incubated with 100 .mu.l/well
of permeabilization buffer (1.times.PBS with 1% Triton-X) with
gentle shaking. The plate was then blocked with 100 .mu.l of 3% w/v
bovine serum albumin for 2 hours at room temperature (RT) and
subsequently washed out of blocking solution with wash buffer
(1.times.PBS with 0.1% Tween-20).
[0730] SARS-CoV-2 viruses on the plate were detected using an
antibody mixture consisting of CC6.29, CC6.33, L25-dP06E11,
CC12.23, CC12.25, which were derived from convalescent SARS-CoV-2
cohort participants (T. F. Rogers et al., Science 369, 956-963
(2020).). Pooled antibodies were added to wells at a concentration
of 2 .mu.g/ml (50 .mu.l/well) and incubated for 2 hours at RT.
Cells were subsequently washed 3 times with wash buffer, stained
with 0.5 .mu.g/ml peroxidase-conjugated AffiniPure goat anti-human
IgG (Jackson ImmunoResearch Laboratories, Inc, Cat #109-035-088)
for 2 hours at RT, and followed by 6 washes with wash buffer. HRP
substrate (Roche, Ca #11582950001), freshly prepared at a 100:1
volume ratio of Solution A:B, was added to each well.
Chemiluminescence was measured using a microplate luminescence
reader (BioTek, Synergy 2).
[0731] A standard curve of serially diluted virus from 3000 to 1
PFU was plotted against relative light units (RLU) using a
4-parameter logistic regression as follows: y=a+(b-a)
(1+(x/x.sub.0).sup.c), where y=variable in RLU, x=variable in PFU
and a, b, c and x.sub.0 are parameters fit by the standard curve.
Using parameters generated by the standard curve, sample RLU values
were converted into PFU values
(x=x.sub.0.times.log.sub.c[(b-y)/(y-a)]), and percentage
neutralization was calculated with the following equation: %
Neutralization=100.times.[(VC-ADI-58124 treated)/(VC-CC), where
VC=average of vehicle-treated control and CC=average of cell only
control, both variables in PFU values. Fifty percent inhibition
titer (IC.sub.50) values were determined using logistic regression
fitting of neutralization curves.
Example 26: Further Neutralization Breadth Evaluation of
Post-Affinity Maturation Antibodies
[0732] Next, the breadth of sarbecovirus recognition by ADI-58124,
ADI-58125, and other clinical antibodies was assessed by measuring
its apparent binding affinity (K.sub.D.sup.App) to a panel of 17
representative sarbecovirus RBDs expressed on the surface of yeast
(T. N. Starr et al., Cell 182, 1295-1310 (2020)). ADI-58125 and
ADI-58124 share the same CDR sequences and only differ in the Fc
region which has been engineered for half-life extension
purpose.
[0733] Thirteen viruses were selected from clade I--representing
the closest known relatives of SARS-CoV-2 (GD-Pangolin and RaTG13)
to the most divergent (SHC014 and Rs4231)--as well as four viruses
from the distantly related clades 2 and 3, which do not utilize
ACE2 as a host receptor (M. Letko, A. Marzi, V. Munster, Nat
Microbiol 5, 562-569 (2020)) (FIG. 40A). Recombinant hACE2 and the
clinical SARS-CoV-2 nAbs described above were also included as
controls. Consistent with previous reports (D. Wrapp et al.,
Science 367, 1260-1263 (2020); T. N. Starr et al., Cell 182,
1295-1310 (2020)), hACE2 only recognized clade I RBDs and bound
with higher affinity to SARS-CoV-2 than SARS-CoV (FIG. 40B). In
addition, the clinical SARS-CoV-2 nAbs CB6/JS016, REGN10987, and
REGN10933 bound to the SARS-CoV-2 RBD with K.sub.D.sup.Apps
comparable to published reports (FIG. 40B) (R. Shi et al., Nature
584, 120-124 (2020).; J. Hansen et al., Science 369, 1010-1014
(2020)). Notably, S309 displayed diminished binding in this
expression platform, likely due to recognition of an epitope
containing an N-glycan that may be hyper-mannosylated in yeast (D.
Pinto et al., Nature 583, 290-295 (2020)).
[0734] Consistent with their broadly neutralizing activities, S309,
ADI-58124, and ADI-58126 displayed remarkably broad binding
reactivity to clade I sarbecovirus RBDs, with ADI-58124 and
ADI-58126 strongly binding 12/13 viruses and S309 binding all 13
(FIG. 40B). In contrast, ADI-58120 bound to 9/13 viruses and
CB6/JS016, REGN10987, and REGN10933 bound only the closest
evolutionary neighbor(s) of SARS-CoV-2, consistent with their
narrow neutralization profiles (FIG. 39B and FIG. 40B). Notably,
ADI-58124 bound with high affinity (KDApp 0.24-1.12 nM) to every
clade I sarbecovirus RBD that exhibited detectable hACE2 binding in
our assay. This finding supports the high degree of ADI-58124
epitope conservation among sarbecoviruses that can utilize hACE2 as
a receptor.
[0735] Several recent studies have shown that RBD mutants that are
resistant to commonly elicited SARS-CoV-2 nAbs are circulating at
low levels in the human population (B. Korber et al., Cell 182,
812-827 (2020).; Y. Weisblum et al., bioRxiv, (2020)). The breadth
of ADI-58124 binding to naturally circulating SARS-CoV-2 variants
that contain single point mutations in the RBD was assessed.
ADI-58120, ADI-58126, and the clinical SARS-CoV-2 nAbs were also
included as comparators. Using the yeast surface-display platform
described above, we expressed the 36 most frequently observed
SARS-CoV-2 RBD variants reported in the GISAID database as well as
several naturally circulating SARS-CoV-2 variants that have been
shown to be resistant to previously described SARS-CoV-2 nAbs (Y.
Shu, J. McCauley, Euro Surveill 22, 30494 (2017).; B. Korber et
al., Cell 182, 812-827 (2020).; Y. Weisblum et al., bioRxiv,
(2020)). One or more of the 36 SARS-CoV-2 variants displayed
resistance (<25% of WT binding) to ADI-58120, CB6/JS016,
REGN10987, and REGN10933. Notably, the resistant variants
identified for REGN10987 and REGN10933 partially overlapped with
those identified in previous in vitro neutralization escape
studies, validating the use of our RBD display platform for the
prediction of antibody escape mutations (A. Baum et al., Science
369, 1014-1018 (2020)). In contrast, ADI-58124, ADI-58126, and S309
bound to all 36 variants with affinities >50% of WT SARS-CoV-2
(FIG. 40C). This result, combined with the remarkable
neutralization breadth observed for these three mAbs (FIG. 39B,
FIG. 40B, and FIG. 40D), suggests a potential link between epitope
conservation and resistance to viral escape.
[0736] Binding breadth for ADI-58125 was determined in the same
manner and the results are provided in FIGS. 40E-40F.
[0737] Unlike other clinical antibodies, ADI-58125 binds to
residues that are not targeted by the endogenous antibody response.
Indeed, other clinical antibodies bind around residues that are
subject to frequent mutations, such as residues 439, 453, 417, 484,
494, 490, 444, 446, or 484 which were shown to have a frequency of
mutations at site of about 102 to 10-, and some of the most
frequent mutations also fall within these regions, for example,
N439K (with a cumulative prevalence of 1.49%), and Y453F (with a
cumulative prevalence of 0.365%) (Greaney et al., Comprehensive
mapping of mutations to the SARS-CoV-2 receptor-binding domain that
affect recognition by polyclonal human serum antibodies, BioRxiv,
2020). For example, other "class 1" antibodies such as C105,
CC12.1, CC12.3, COVA2-4, B38, Ly-Cov16, and REGN10933 bind to
residues around Q493R, Y453F, F486V, and K417N. Other "class 2"
antibodies such as C104, P2B-2F6, BD23, Ab2-4, 5A6 and Ly-CoV555
bind to residues around E484K, F490L, and S494P. Other "class 3"
antibodies such as C135 and REGN10987 bind around N440K, K444N,
V445E, N439S, and G446V. Finally, other "class 4" antibodies such
as CR3022 and EY6A bind to different regions/residues. However, all
these regions with a high frequency of mutations occurred outside
the binding epitope for ADI-58125. Other mutations include S477N
(with a cumulative presence of 5.69%, N501Y (with a cumulative
presence of 1.39%), E484K, K417N, S494P, L452R, G446V, F490S,
L452M, L455F, E484Q, F486L and G485R (Greaney et al., BioRxiv,
2020).
[0738] Furthermore, ADI-58125 was able to bind to all common
circulating SARS-CoV-2 variants described to date, such as the UK
lineage, the South Africa lineage, the newly emerged B.1.1.7
lineage and/or the mink strain (FIG. 40G). The B.1.1.7 variant
carries a large number of mutations, suggesting that it may have
arisen in a chronically infected patient (Kemp et al., Recurrent
emergence and transmission of a SARS-COV-2 spike deletion H69/H70,
BioRxiv, 2020; Gupta et al., Biorxiv, 2020; the entire contents of
each of which are expressly incorporated herein by reference).
Several of the mutations in this variant are in the S protein
including a deletion at positions 69 and 70 that evolved
spontaneously in other SARS-CoV-2 variants and is hypothesized to
increase transmissibility (KempSA et al., BioRxiv 2021). This
disproportionate number of mutations in the S protein strongly
suggests immune escape. The NTD deletion often co-occurs with RBD
mutations N501Y, Y453F, N439K, and E484K and K417N.
[0739] ADI-58125 was able to bind with high affinity to 12 of 13
clade 1 sarbecoviruses, including all RBDs exhibiting detectable
human ACE2 binding. RBDs harbouring mutations present in the
B.1.351 and P.1 variants, which are associated with increased
resistance to neutralisation by convalescent and vaccinee sera,
bound with reduced affinity to several clinical antibodies (FIG.
40H). In contrast, ADI-58125 retained high affinity binding to all
common SARS-CoV-2 variants as well as to the B.1.1.7, B.1.351 and
P.1 variants (FIG. 40I). Similarly, ADI-58122 and ADI-58127 also
retained high affinity binding to P.1 strain. Indeed, although the
variants can escape neutralization by a number of monoclonal
antibodies, these three antibodies (ADI-58122, ADI-58125 and
ADI-58127) showed little to no reduction in neutralization activity
against the varaints, and also neutralized P.1 varaint with all
reaching a plateau at 100% neutralization, whereas REGN10987 and
S309 failed to reach 100% neutralization (FIG. 40I and FIG. 50C).
Authentic B.1.1.7 and B.1.351 variants remained fully susceptible
to ADI-58125 neutralisation (data not shown).
[0740] ADI-58125 displays exceptional breadth of binding to RBDs
from clade 1 sarbecoviruses and variants of SARS-CoV-2 resistant to
other antibody therapies. No loss of binding affinity for the
B.1.1.7, B.1.351 and P.1 variants was observed, and ADI-58125
neutralisation potency was maintained against the B.1.1.7 and
B.1.351 variants. The unique, broadly neutralising activity of
ADI-58125 highlights its potential to be an effective prophylactic
and therapeutic agent against emergent variants of SARS-CoV-2
resistant to other clinical stage mAbs as well as pre-emergent
SARS-like viruses with pandemic potential.
[0741] ACE2 Inhibition of S by ELISA
[0742] The median inhibitory concentration (IC.sub.50) of ADI-58125
required to block binding of spike protein to ACE2 was measured in
a competition ELISA assay. ADI-58125 potently blocked ACE2 binding
with a sub-nanomolar IC.sub.50 of 0.022 mM (3.3 ng/ml) (FIG. 40J).
REGN10933, a known ACE2 competitor, blocked S binding, while S309,
a known non-competitor, showed minimal inhibition of S binding,
thus validating the use of this assay. ADI-58125's strong blocking
activity is consistent with its high binding affinity and supports
ACE2 blocking as a primary mechanism of its potentneutralization
against SARS-CoV-2.
[0743] Brief description of methods used is provided below.
[0744] Sarbecovirus Phylogeny and Alignment:
[0745] Representative sarbecovirus RBD-SD1 sequences were selected
based on sequence sets curated by Letko et al. and Starr et al.
(Letko M. et al., Nat Microbiol 5, 562-569 (2020); T. N. Starr et
al., Cell 182, 1295-1310 (2020)). Four other ACE2-utilizing
sarbecoviruses (Frankfurt 1, CS24, Civet 007-2004, and A021) not
studied in previously curated sets were added here as each
possessed unique sequences at the RBD-ACE2 interface, thus spanning
additional sequence distance across the clade I phylogeny. A
limited set of clade 2 and clade 3 members, which are known to use
alternative receptors to ACE2, were included as controls (Letko M.
et al., Nat Microbiol 5, 562-569 (2020)). A phylogram of
sarbecoviruses was generated using maximum likelihood analysis of
mafft-aligned RBD-SD1 sequences (FIG. 40A). Multiple sequence
alignment of sarbecovirus RBD sequences was visualized in Jalview
(FIG. 41J). Amino acid sequences for each sarbecovirus was colored
by percentage sequence identity and overall degree of conservation
per residue was calculated as a numerical index weighted by
physio-chemical properties of amino acids (Livingstone C. D. et
al., Comput Appl Biosci. 1993 December; 9(6):745-56).
[0746] GISAID Analysis of Circulating SARS-CoV-2 Variants:
[0747] Genome sequences were downloaded from the GISAID database
and pairwise aligned against the reference Wuhan-Hu-1 sequence (ENA
QHD43416.1) via an internal implementation of the Needleman-Wunsch
algorithm to extract all RBD-SD1 sequences using amino acid
residues 319 to 591 of the Wuhan-Hu-1 spike sequence. Incomplete
RBD-SD1 nucleotide sequences and those containing ambiguous ("n")
base calls, plus translated sequences including "X", "*", or "-,"
were excluded from analysis. RBD-SD1 sequence variants observed at
least 6 times out of 63551 sequences analyzed as of Jul. 14, 2020,
as well as several literature controls and antibody escape mutants
also observed in the GISAID database, were compiled as a panel 36
variants to assess antibody binding.
[0748] As the number of sequences in the GISAID database has been
consistently growing since the start of the COVID-19 pandemic,
sequences from the GISAID database were pulled again on Oct. 19,
2020 to calculate the "percent prevalence" of each variant.
"Percent prevalence" was calculated by dividing the number of
appearances of the variant by the total number of complete
sequences in the database.
[0749] SARS-CoV-2 Variants and Homologous Sarbecovirus RBD-SD1
Cloning:
[0750] The spike RBD-SD1 of SARS-CoV-2 (residues 319 to 591 as
defined by Uniprot: P0DTC2) and additional related sarbecoviruses
(HKU3, ENA AAY88866.1; Rfl-2004, ENA ABD75323.1; BM48-31, ENA
ADK66841; Pangolin_GX-P2V GISAID MT072864.1; RaTG13, ENA
QHR63300.2; SARS-CoV-2, ENA QHD43416.1; GD-Pangolin, ENA
MT121216.1; Rs4231, ENA ATO98157.1; WIV1, ENA AGZ48831.1; Civet
007-2004, ENA AAU04646.1; A021, ENA AAV97986.1; Frankfurt 1, ENA
BAE93401.1; SARS-CoV-1, ENA AAP13441; CS24, ENA ABF68959; LYRa11,
ENA AHX37558.1; Rs4081, ENA KY417143.1) were ordered as gBlocks
(IDT) and cloned into a yeast display expression vector encoding a
flexible Gly4Ser linker (SEQ ID NO: 23194) and hemagglutinin (HA)
tag at the N-terminus. Two consecutive Gly4Ser linkers (SEQ ID NO:
23195) connect RBD-SD1 to Aga2p at the C-terminus. 36 circulating
SARS-CoV-2 variant sequences observed in the GISAID database
(T3231, P330S, V3411, A344S, N354D, S359N, V367F, N370S, F377L,
V382L, P384L, P384S, R403K, R4081, Q414R, N439K, N440K, K444N,
G446V, Y453F, A475V, G476S, S477N, T4781, P479S, V483A, E484D,
E484K, F490L, F490S, Q493R, S494P, N501Y, A520S, A522V, A522S) were
inserted into the same vector described above. The A352S variant
was excluded due to an error present in the gBlock. Plasmids were
transformed into S. cerevisiae (EBY100) using the Frozen-EZ Yeast
Transformation II Kit (Zymo Research) following the manufacturer's
protocol and selected via the tryptophan auxotrophic marker.
[0751] Yeast-Surface Display of RBD-SD1:
[0752] For induction of RBD expression, fresh yeast cultures were
inoculated at an OD.sub.600 density of 0.2 in selective SDCAA media
and grown at 30.degree. C. and 180 rpm until cultures reached an
OD.sub.600 of 0.8 to 1.0. Next, cells were centrifuged at
2400.times.g for 3 minutes, resuspended in an equal volume of SGCAA
(6.7 g/L Yeast Nitrogen Base, 4.0 g/L drop out amino acid mix, 0.46
g/L NaH.sub.2PO.sub.4, 0.88 g/L Na.sub.2HPO.sub.4, 7.7 g/L NaCl, 2%
galactose, 2% raffinose), and incubated for 16 to 20 hours at
20.degree. C., 200 rpm.
[0753] Antibody Binding to Yeast-Displayed RBD Variants:
[0754] To assess binding breadth, IgGs and hACE2 (expressed in a
bivalent format as a C-terminal IgG1 Fc conjugate; Sino Biological,
Cat #10108-H02H) were tested against the panel of 17 sarbecovirus
RBDs. Initially, binding was determined at a single 100 nM
concentration of IgG or hACE2. Briefly, 0.2 OD induced cells per
well were aliquoted into 96-well plates and washed out of SGCAA
media with PBSF. Next, cells were resuspended in 100 .mu.l of 100
nM IgG or hACE2 and incubated at room temperature for 30 minutes.
Cells were subsequently washed twice with PBSF and labeled with 50
.mu.l of allophycocyanin (APC)-conjugated monoclonal mouse
anti-hemagglutinin tag (HA).11 antibody (BioLegend, Cat #901524),
phycoerythrin (PE)-conjugated goat anti-human IgG polyclonal
antibodies (Southern Biotech, Cat #2040-09), and propidium iodide
(Invitrogen, Cat #P1304MP) for 20 minutes on ice. For each
sarbecovirus RBD, a secondary reagent control was included. Cells
were washed twice with PBSF before analyzing via flow cytometry on
a BD FACS Canto II (BD Biosciences).
[0755] To account for differences in RBD expression across
sarbecoviruses, binding signal was normalized to HA-tag signals
(MFI.sub.anti-human IgG PE/MFI.sub.anti-HA APC). Binding with
normalized ratios below 1.0 were considered non-binding (NB) at the
concentration tested. Those with ratios above 1.0 were titrated to
calculate their apparent binding affinity (K.sub.D.sup.App).
[0756] Titrations were performed as 2-fold dilution series from 100
nM to 0.048 nM IgG or hACE2 as described above to obtain
K.sub.D.sup.App values. Mean anti-human IgG PE MFI signal was
normalized from 0 to 100 (MFI.sub.[ADI-58124 or hACE2
concentration]-MFI.sub.minimum)*100/(1-MFI.sub.minimum) and fitted
as nonlinear regression curves in GraphPad Prism using the
following equation:
Y=Y.sub.x=min+X*(Y.sub.x=max-Y.sub.x=min)/(K.sub.D.sup.App+X),
where X is the IgG or hACE2 concentration and Y is the normalized
binding signal. Points displaying hook effects, defined as PE MFI
collected at concentrations higher than that of the maximum MFI
concentration, were excluded from analysis. K.sub.D.sup.App (nM)
for inventive antibodies and clinical antibodies and hACE2 are
displayed as a heatmap (FIG. 40B).
[0757] To maximize the dynamic range of potential differences in
binding affinity to SARS-CoV-2 variants, binding experiments were
conducted at each antibody's respective SARS-CoV-2 K.sub.D.sup.App.
Binding signal was normalized using the following equation:
MFI.sub.anti-hu IgG PE/MFI.sub.anti-HA APC-MFI.sub.background
anti-hu IgG PE/MFI.sub.background anti-HA APC, and calculated as a
percentage of normalized signal of the reference WT SARS-CoV-2
strain RBD-SD1.
Example 27: Further Analyses on Antigen Recognition by
Post-Affinity Maturation Antibodies
[0758] First, to gain further insight into the antigenic surface
recognized by ADI-58124, a mutagenized yeast surface-display RBD
library was generated and rounds of selection were performed to
identify RBD variants that displayed loss of binding to ADI-58124
(FIGS. 41A-41C). A final round of positive selection was performed
using a mixture of recombinant hACE2 and two RBD-directed mAbs
(S309 and CR3022) that target non-overlapping epitopes distinct
from the ADI-58124 binding site to exclude mutations that globally
disrupt the conformation of the RBD (D. Pinto et al., Nature 583,
290-295 (2020).; M. Yuan et al., Science 368, 630-633 (2020).).
Selected RBD mutants encoding single amino acid substitutions were
individually tested for binding to ADI-58124, recombinant hACE2,
CR3022, and S309 to confirm site-specific knock-down mutations
(FIGS. 41B and 41D). Substitutions at only four RBD positions
specifically abrogated ADI-58124 binding: D405E, G502E/R/V,
G504A/D/R/S/V and Y505C/N/S (FIGS. 41E-41F). These four residues
are remarkably conserved among the clade I sarbecovirus subgenus
and invariant among SARS-CoV-1, SARS-CoV-2, SHC014 and WIV1 viruses
(FIG. 41G), providing a molecular explanation for the breadth of
binding and neutralization exhibited by ADI-58124. Consistent with
the conservation of these residues among clade I sarbecoviruses,
none of the substitutions that impacted ADI-58124 binding were
present in full-length SARS-CoV-2 sequences deposited in the GISAID
database as of Oct. 19, 2020. Notably, 3 of the 4 identified
mutations that abrogate ADI-58124 binding lie within the hACE2
binding site (Lan J. et al., Nature. 2020 May; 581(7807):215-220.))
and at least one mutation at each position (G502E/R/V, G504V and
Y505C/N/S) also abrogated hACE2 binding (FIGS. 41E-41F), likely
accounting for their absence among circulating SARS-CoV-2 isolates.
These results suggest that the evolutionary conservation of the
ADI-58124 epitope is likely directly linked to ACE2 binding.
[0759] Next, to further understand the epitope of ADI-58120,
ADI-58124, ADI-58126, ADI-58128, and ADI-58130, antibody-resistant
SARS-CoV-2 S protein was induced using recombinant vesicular
stomatitis virus encoding the SARS-CoV-2 S protein
(rVSV-SARS-CoV-2-S, from Wuhan-Hu-1 isolate) as a surrogate
recombinant screening system. Pre-titrated rVSV-SARS-CoV-2-S was
mixed with serially diluted amounts of test antibodies and
incubated with Vero cells and repeatedly passaged. Neutralization
assay was performed with the potentially resistant virus obtained
from the final passage.
[0760] Neutralization of mutant SARS-CoV-2 S-comprising pseudovirus
(rVSV) by ADI-58120, ADI-58124, ADI-58126, ADI-58128, and ADI-58130
are provided in FIGS. 41H-41J. Based on the increase in the IC50
values shown in FIGS. 41H-41J, N440H and N440D substitutions in the
S protein independently conferred resistance to neutralization by
ADI-58120, G504D and G504S substitutions independently conferred
resistance to neutralization by ADI-58124, T415I substitution
conferred resistance to neutralization by ADI-58126, F490S
substitution conferred resistance to neutralization by ADI-58128,
and Y145D, K150E, and W152R substitutions independently conferred
resistance to neutralization by ADI-58130. Accordingly, residue 440
is a proposed epitope residue for ADI-58120, residue 504 is a
proposed epitope residue for ADI-58124, residue 415 is a proposed
epitope residue for ADI-58126, residue 490 is a proposed epitope
residue for ADI-58128, and residues 145, 150, and 152 are proposed
epitope residues for ADI-58130.
[0761] Methods used for the RBD yeast library-based study and the
antibody-resistant S protein selection study for ADI-58124 are
provided below. Essentially the same methods were used for
ADI-58120, ADI-58126, ADI-58128, and ADI-58130.
[0762] ePCR Library Construction and Selection of RBD Mutants:
[0763] SARS-COV-2 RBD-SD1 gBlock (IDT) was amplified by polymerase
chain reaction (PCR) with iProof High-Fidelity PCR system (Bio-Rad,
Cat #1725310) following the manufacturer's recommendations. The
amplified DNA was purified (Nucleospin Gel and PCR Clean-up Kit,
Macherey-Nagel, Cat #740609.250) and subsequently mutagenized by
error-prone PCR(ePCR) using the GeneMorph II Random Mutagenesis Kit
(Agilent Technologies, Cat #200550) with a target nucleotide
mutation frequency of 0-4.5 mutations per kilobase of DNA. The
mutagenized DNA product was cloned into yeast via electroporation
as described earlier. The ePCR library was validated by plating a
subset of the transformed ePCR yeast library on tryptophan dropout
agar plates (Teknova, Cat #C6099) and Sanger sequencing clonal
yeast cells. The WT SARS-CoV-2 RBD-SD1, cloned as described
earlier, was used as a reference in subsequent selection
efforts.
[0764] Prior to performing FACS selection, ePCR RBD-SD1 library and
WT RBD-SD1 yeast were induced as described above. To select for
mutants with diminished binding to ADI-58124, yeast cells were
stained with ADI-58124 in a similar format as described above.
Briefly, induced cells were incubated for 30 minutes on ice with
ADI-58124 IgG diluted in PBSF to its EC.sub.80 concentration, which
was determined by titrating ADI-58124 on yeast-displayed WT RBD-SD1
(FIGS. 41A-41D). Cells were then washed twice in PBSF, stained in a
secondary staining mixture and analyzed on a BD FACS Aria II
(Becton Dickerson). A subset of yeast population exhibiting HA-tag
expression but reduced ADI-58124 binding relative to WT RBD-SD1
yeast, as shown in FIG. 40A, were sorted and propagated in SDCAA
media for 48 hours at 30.degree. C. This selection procedure was
repeated for a second round to further enrich yeast encoding
ADI-58124 binding knock-down mutations. In the final round of
selection, the induced library was stained with a mixture of hACE2,
S309 and CR3022 at their respective EC.sub.80 concentrations. The
subset of the stained population that mirrored the binding profile
of WT RBD-SD1-stained yeast was sorted and plated on agar plates
for Sanger sequencing of single colonies. Individual clones
possessing single amino acid substitutions identified from
sequencing were cultured, induced, and evaluated for binding to
ADI-58124, S309, CR3022 and soluble hACE2 at their respective
EC.sub.80 concentrations through flow cytometric analysis on the BD
FACS Canto II (BD Biosciences). Binding signal was normalized and
calculated as a percentage of the binding signal to reference WT
RBD-SD1, as described earlier.
[0765] Mutant SARS-CoV-2 S-Comprising Pseudovirus rVSV Escape
Study:
[0766] The concentration that gives 90% of the maximum inhibition
(IC90) for ADI-58124 was estimated in a 9-point neutralization
assay. Briefly, a pre-titrated amount of rVSV-SARS2 S virus was
incubated with serial dilutions of ADI-58124 for 1 hour at room
temperature. For screening the antibody-virus mixture was applied
to monolayers of Vero cells in a 96-well plate. Following a 7-hour
incubation, eGFP-positive virus-infected cells were enumerated
using a Cytation-5 imager (Biotek) and analyzed with the onboard
Gen5 software, version 3.04 (Biotek).
[0767] Vero cells were plated in a 12-well plate the day before
selection with the antibody so that cells would reach .about.80%
confluency the next day. Parental rVSV-SARS-CoV-2 S virus was
titrated to obtain a viral infection of .about.2% in the 12-well
plate at 8 hours after infection. The next day, ADI-58124 was
incubated at the estimated IC90 with different multiplicity of
infections (increasing by 3-fold for 3 different treatments) of the
rVSV-SARS-CoV-2 S virus at room temperature for 1 hour. The same 3
concentrations of virus were also tested in wells with no
ADI-58124, but vehicle, phosphate buffered saline, as controls.
Vero cells were infected with the virus and ADI-58124 mixtures. The
plates were monitored for signs of eGFP expression at 8 to 10 hours
after infection and every 12 hours thereafter. The virus was
harvested approximately 2 to 3 days after infection, when most of
the cells were infected in the ADI-58124-treated wells. The
supernatant was centrifuged at 15,000 rpm for 1 minute at 4.degree.
C. to get rid of cell debris, then aliquoted and stored at
80.degree. C.; the virus harvested was Passage 1 (P1) of the
selection with ADI-58124 to find potential antibody-resistant
rVSV-SARS-CoV-2 S virus.
For P2, virus from the well with lowest virus inoculum was
incubated with twice the concentration of ADI-58124 selected for
the assay for P1. The mixtures were incubated and then added to the
Vero cells and harvested as described for P1. The selection
protocol was repeated until 3 passages were conducted, at which
point a neutralization assay to compare parental virus and the
potentially resistant virus population to estimate resistance to
ADI-58124 was performed. Single viral clones were plaque-purified
when the viral population shows a shift of 10-fold or more in the
IC50 for neutralization of ADI-58124. The plaque-purified virus was
amplified on Vero cells in the presence of an IC90 concentration of
antibody to prevent any potential reversion to the parental
genotype. Purified viral clones were incubated with ADI-58124 to
verify resistance to the antibody, as compared to the parental
virus in a neutralization assay as described above. RNA was
extracted from 700 .mu.L supernatant of resistant clones using
RNeasy Mini Kit (Qiagen, Cat. No. 74136) as per manufacturer's
instructions. The S gene was amplified by RT-PCR using the
SuperScript.RTM. III First-Strand Synthesis Kit (Invitrogen, Cat.
No. 18080051) and PCR products were gel-purified by using the
QIAQuick Gel Extraction Kit (Qiagen, Cat. No. 28706) following the
manufacturer's protocols. Gel-purified PCR product was sent out to
Genewiz (South Plainfield, N.J.) for Sanger sequencing to identify
the genotype.
Example 28: SARS-CoV-2-S Binding Competition Analyses
[0768] To further understand the epitope of the novel antibodies,
competitive binding analyses were conducted using BLI. Competition
between ACE2 and respective antibodies for binding to SARS-CoV-2-S
was first analysed. As shown in FIGS. 42A-42B, ADI-58120,
ADI-58121, ADI-58122, ADI-58123, ADI-58124, ADI-58125, ADI-58126,
ADI-58127, ADI-58128, and ADI-58129 competed with ACE2, while
ADI-58130 and ADI-58131 did not. All tested clinical antibodies
except S309 competed with ACE2. Competition between two different
antibodies were also analyzed. Results are shown in FIGS.
42C-42G.
[0769] Based on these results, competition between two antibodies
and competition between hACE2 and an antibody are summarized in
FIG. 42H. Table 4 provides the framework region mutations in VH and
VL chain of the selected antibodies.
TABLE-US-00004 TABLE 4 VH VH VL VL Non-Designed Designed
Non-Designed Designed FR FR FR FR ADI ID Mutations mutations
Mutations mutations ADI-58120 5 0 4 0 ADI-58121 5 0 4 0 ADI-58122 5
0 4 0 ADI-58123 5 0 4 0 ADI-58124 2 0 1 0 ADI-58125 2 0 1 0
ADI-58126 3 0 0 0 ADI-58127 3 0 0 0 ADI-58128 2 0 0 0 ADI-58129 2 0
0 0 ADI-58130 0 0 0 0 ADI-58131 0 0 0 0
[0770] Methods used for the competitive binding studies are
provided below.
[0771] ACE2 Competitive Binding Studies:
[0772] Biosensor Instrument, Sensor Tip, Assay Buffer and Assay
Conditions: BLI analysis was conducted at 25.degree. C. in a PBSF
buffer system using a ForteBio Octet HTX (Sartorius Bioanalytical
Instruments, Bohemia, N.Y.) equipped with AHC sensor tips.
[0773] Reagent Preparation: The SARS-CoV-2 S and ACE2 proteins were
prepared in bulk by dilution of the stock solution with PBSF buffer
to a concentration of 100 nM.
[0774] Antibody Formulation: The antibodies were diluted from their
stock concentrations to 100 nM.
[0775] Sensor Tip Preparation: The AHC sensor tips were soaked in
PBSF buffer for 10 minutes and then exposed (.about.60 s) to wells
containing the IgGs. The loaded sensors were then soaked in PBSF
buffer for 15 min. Any remaining Fc capture sites on the sensor tip
were blocked by exposing (10 min) the IgG loaded sensor tips with
an irrelevant IgG (adalimumab, 0.5 mg/mL). These loaded and blocked
sensor tips were soaked in PBSF buffer for 30 minutes before
proceeding to the competitive binding experiment.
[0776] Experiment Steps: Each experiment cycle began with dipping
(180 s) the sensor tip into PBSF buffer to establish a stable
baseline. This was followed by exposure (180 s) of the IgG loaded
sensor tip to wells containing ACE2. This step is necessary to show
whether there is any interaction between the IgG and ACE2. After a
short dip (60 s) into fresh wells of PBSF buffer, the sensor tip
was dipped (180 s) into wells containing the SARS-CoV-2 S protein.
The sensor tips were then immediately dipped (180 s) into wells
containing ACE2 protein to monitor any association of ACE2 to
antibody bound SARS-CoV-2 S protein.
[0777] Data Processing: The data was cropped to isolate the
SARS-CoV-2 S and ACE2 exposure steps and then x and y-axis aligned
using ForteBio Data Analysis software version 11.1.3.10.
[0778] Competitive Binding Experiments Using
Biolayer-Interferometry:
[0779] Competition between antibodies for binding to soluble
SARS-CoV-2 S protein was assessed using the ForteBio Octet HTX
(Sartorius Bioanalytical Instruments). All reagents were diluted to
100 nM in PBSF. AHC sensor tips were loaded with ADI-58124 IgG,
followed by exposure to an inert IgG to block any remaining Fc
capture sites. Tips were subsequently equilibrated in PBSF for 30
min. ADI-58124-loaded sensor tips were transferred to wells
containing hACE2, CR3022, or S309 to check for any interaction with
ADI-58124. Sensor tips were then loaded in wells containing fresh
PBSF buffer (60 s), followed by exposure to SARS-CoV-2 S protein
(180 s), and lastly, exposure to hACE2, CR3022, or S309 (180 s).
Data were cropped to include only SARS-CoV-2 S protein and hACE2,
CR3022, or S309 exposure steps and aligned by x- and y-axes using
ForteBio Data Analysis software version 11.1.3.10.
Example 29: Fc-Mediated Effector Functions by Post-Affinity
Maturation Antibodies
[0780] ADI-58125 and ADI-58124 share the same CDR sequences and
only differ in the Fc region which has been engineered for
half-life extension purpose. Because Fc-mediated effector functions
can contribute to protection independently of viral neutralization,
ADI-58124 and ADI-58125 binding to different Fc gamma receptors
(FcgRs), neonatal Fc receptor (FcRn), and the complement component
C1q was tested. Results are shown in FIGS. 43A-43C.
[0781] FIG. 43A shows that there is no significant difference in
binding of any of these tested FcgR proteins to ADI-58124 and
ADI-58125. This indicates suggesting that the Fc half-life
extension LA mutations in ADI-58124 do not affect binding to any of
the FcgR proteins assessed in the studies here. Functionally, this
implies that ADI-58125 will possess normal effector functions
mediated through interaction with FcgR, which may contribute to
ADI-58125 SARS-CoV-2 neutralizing activity.
[0782] FIG. 43B shows that at pH 6.0, the ADI-58124 and ADI-58125
Fc variants provide slightly different binding profiles to human
and cyno FcRn. In general, each IgG binds to cyno FcRn slightly
stronger than to human FcRn. For both human and cyno FcRn,
ADI-58125 (LA Fc) bound with higher affinity than ADI-58124 (WT Fc)
at pH 6.0. The enhanced affinity of ADI-58125 to FcRn at pH 6.0 is
expected to translate to extended in vivo half-life. Neither
ADI-58124 nor ADI-58125 bound to FcRn at pH 7.4.
[0783] FIG. 43C shows that ADI-58124 and ADI-58125 have equivalent
affinities to C1q and, therefore, the LA mutations in ADI-58125 are
not expected to impact IgG1 Fc-mediated complement pathway
activation.
[0784] Methods used to assess Fc binding are provided below.
[0785] FcgR Binding Studies:
[0786] Biosensor Instrument, Sensor Chip, Running Buffer and Assay
Conditions: SPR analysis was conducted at 25.degree. C. in a
HBS-EP+ buffer system (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA,
0.05% Surfactant P20) using a Biacore 8K optical biosensor equipped
with a CAP sensor chip (Global Life Sciences Solutions USA,
Marlborough, Mass.). (Global Life Sciences Solutions USA,
Marlborough, Mass.). The sample compartment was maintained at
10.degree. C. for the duration of the experiment. This assay
orientation allows for reproducible capture of biotinylated samples
to the sensor surface.
[0787] Surface Preparation: Prior to each analysis the sensor chip
surface was first conditioned with 3 pulses (60 s at 10 .mu.L/min)
of regeneration solution (6 M Guanidine-HCl in 0.25 M NaOH).
[0788] Antigen Preparation: SARS-CoV-1 RBD was prepared in bulk by
dilution of the stock samples with HBS-EP+ buffer at a test
concentration of 6.0 nM.
[0789] ADI-58124 and ADI-58125 Formulation: Both antibodies were
diluted from their stock concentrations to a concentration of 27.0
nM.
[0790] FcgR Formulation: The FcgRI protein was diluted from its
stock concentration to 32 nM and then serially diluted (2-fold) to
a concentration of 1.0 nM. All other FcgR proteins were diluted
from their stock concentrations to 1024 nM and then serially
diluted (2-fold) to a concentration of 8.0 nM
[0791] Experiment Steps: Each experiment cycle began with an
injection (300 s at 2 .mu.uL/min) over flow cells 1 and 2 of a 1:20
solution of biotin CAPture reagent (Global Life Sciences Solutions
USA, lot #10275955) in HBS-EP+ buffer. This was followed by an
injection (180 s at 10 .mu.L/min) of the biotinylated antigen
overflow cell 2. Upon capture of the antigen to the sensor surface,
ADI-58124 or ADI-58125 was injected (180 s at 30 .mu.l/min)
overflow cells 1 and 2. The dissociation of the IgG was monitored
for 120 s prior to injection (180 s) of the FcgR. Dissociation of
the FcgR from the sensor surface was monitored for 180 s. Finally,
an injection (120 s at 10 .mu.L/min) of regeneration solution
overflow cells 1 and 2 prepares the sensor surface for another
cycle.
[0792] Model/Fit: The data was first cropped to include only the
steps that involve the FcgR association and dissociation. This
selected data was then aligned, double reference subtracted, and
then non-linear least squares fit to a 1:1 binding model using
Biacore Insight Evaluation software version 3.0.11.15423.
[0793] FcRn Binding Studies:
[0794] Biosensor Instrument, Sensor Chip, Running Buffer and Assay
Conditions: SPR analysis was conducted at 25.degree. C. using a
Biacore 8K optical biosensor equipped with either a CM3 or CM5
sensor chip (Global Life Sciences Solutions USA, Marlborough,
Mass.). The sample compartment was maintained at 10.degree. C. for
the duration of the experiment.
[0795] Instrument Running Buffer: These studies were conducted in
an HBS-EP+ buffer system (10 mM HEPES, 150 mM NaCl, 3 mM EDTA,
0.05% Surfactant PS20) at pH 6.0 or pH 7.4.
[0796] Antibody Formulation: A pH scouting study helped determine
the buffer pH and approximate concentration for direct
immobilization of each IgG to the sensor surface.
[0797] Surface Preparation: The sensor surface was prepared as
follows: a 1:1 mixture of EDC and NHS was injected (420 s) over
flow cells 1 and 2, the antibody was injected (120 s) over flow
cell 2 and finally ethanolamine was injected (420 s) over flow
cells 1 and 2.
[0798] FcRn Formulation: For the experiments performed at a buffer
pH of 6.0, human and cyno FcRn were diluted from their stock
concentrations with HBS-EP+ buffer (pH 6.0) to a concentration of
60.0 nM and serially diluted (2-fold) to 0.029 nM. For the
experiments performed at a buffer pH of 7.4, human and cyno FcRn
were diluted from their stock concentrations with HBS-EP+ buffer
(pH 7.4) to a concentration of 128 nM and serially diluted (2-fold)
to 1.0 nM.
[0799] Experiment Steps: Each experiment cycle began with an
injection (180 s at 30 .mu.uL/min) of FcRn over flow cells 1 and 2.
The dissociation of the FcRn was observed for 180-300 s before the
sensor surface was regenerated via two injections (20 s at 30
.mu.uL/min) of HBS-EP+ buffer (pH 7.4) which prepares the sensor
surface for another cycle.
[0800] Model/Fit: The data was aligned, double reference
subtracted, and then non-linear least squares fit to a 1:1 binding
model using Biacore Insight Evaluation software version
3.0.11.15423.
[0801] C1q Binding Studies:
[0802] Biosensor Instrument, Sensor Tip, Assay Buffer and Assay
Conditions: BLI analysis was conducted at 25.degree. C. in a PBSF
buffer using a ForteBio Octet HTX (Sartorius Bioanalytical
Instruments, Bohemia, N.Y.) equipped with SA sensor tips. These
assay conditions were adapted from Zhou et al. (2)
[0803] Antigen Preparation: Biotinylated SARS-CoV RBD was prepared
in bulk by dilution of the stock sample with PBSF buffer to a
loading concentration of 100 nM.
[0804] Sensor Tip Preparation: The sensor tips were soaked in PBSF
buffer for 10 minutes and then exposed (300 s) to wells containing
biotinylated SARS-CoV RBD. Following an additional 20 minute
incubation in PBSF, the antigen loaded sensors were exposed (90 s)
to wells containing the IgG.
[0805] Antibody Formulation: ADI-58124 and ADI-58125 were diluted
from their stock concentrations to 100 nM.
[0806] C1q formulation: The stock solution of C1q was diluted into
PBSF buffer to 10 nM and then serially diluted (2-fold) to a
concentration of 0.625 nM.
[0807] Experiment Steps: Each experiment cycle began with dipping
(60 s) the sensor tips into PBSF to establish a stable baseline.
The sensor tips were then dipped (180 s) into wells containing C1q
or blank buffer. The sensor tips were then immediately dipped (180
s) into fresh wells containing PBSF buffer to monitor (initial 30
s) the dissociation of C1q from the sensor tip surface.
[0808] Model/Fit: The data was x and y-axis aligned and then
non-linear least squares fit to a 1:1 binding model using ForteBio
Data Analysis software version 11.1.3.10.
[0809] Subsequently, the ability of ADI-58124 and ADI-58125 to
induce antibody-dependent natural killer cell activation and
degranulation (ADNKDA), antibody-dependent cell phagocytosis
mediated by monocytes and neutrophils (ADCP and ADNP), and
antibody-mediated complement deposition (ADCD) was assessed using
previously described in vitro assays (B. M. Gunn et al., Cell Host
Microbe 24, 221-233 (2018)). Clinical SARS-CoV-2 nAbs S309 and
REGN10987 were also included as comparators. ADI-58124 and
ADI-58125 displayed a highly polyfunctional profile, resulting in
the induction of phagocytosis by monocytes and neutrophils,
deposition of the complement component C3, and induction of NK cell
degranulation (a surrogate marker of ADCC) and activation (FIGS.
43D-43E). Interestingly, while ADI-58124, ADI-58125, S309, and
REGN10957 showed comparable recruitment of phagocytosis, these
antibodies differed with respect to complement deposition and NK
cell activation; S309 showed reduced complement deposition compared
with ADI-58124, ADI-58125, and REGN10987, and ADI-58124 and
ADI-58125 showed superior NK cell activation over both S309 and
REGN10987 (FIGS. 43D-43E). In summary, ADI-58124 and ADI-58125
robustly triggers diverse Fc-mediated effector activities with
potencies comparable to or superior than that of current lead
SARS-CoV-2 clinical antibodies.
[0810] Methods used in this example are briefly described
below.
[0811] Ab-Dependent Natural Killer Cell Activation and
Degranulation (ADNKDA):
[0812] Primary human NK cells were enriched from the peripheral
blood of human donors using RosetteSep Human NK cell Enrichment
Cocktail (Stem Cell Technologies, Cat #15065) and cultured
overnight in RPMI-1640 (Corning, Cat #15-040-CV) supplemented with
10% FBS (Hyclone SH30071.03), 1% Pen/Strep (Gibco, Cat #15070-063),
1% L-Glutamine (Corning Cat #25-005-CI), 1% HEPES (Corning, Cat
#25-060-CI) and 5 ng/ml of recombinant human IL-15 (StemCell
Technologies Cat #78031). Recombinant SARS-CoV-2 receptor binding
domain was coated onto MaxiSorp 96-well plates (Thermo Scientific,
Cat #442404) at 200 ng/well at 4.degree. C. overnight. Wells were
washed with PBS and blocked with 5% BSA prior to addition of
antibodies that were diluted in a five-fold dilution series in PBS
(10 .mu.g/ml-0.32 ng/ml) and incubation for 2 h at 37.degree. C.
Unbound antibodies were removed by washing with PBS were added at
5.times.10.sup.4 cells/well in the presence of 4 gg/ml brefeldin A
(Biolegend, Cat #420601), 5 .mu.g/ml GolgiStop (BD Biosciences Cat
#554724) and anti-CD107a antibody (Clone H4A3 PE-Cy7, Biolegend Cat
#328618) for 5 hours. Cells were stained for surface expression of
CD16 (Clone 3G8 Pacific Blue, Biolegend Cat #302032), CD56 (clone
5.1H11 AlexaFluor488 Biolegend, Cat #362518) and CD3 (clone UCHT1
Alexa Fluor700, Biolegend cat #300424). Cells were fixed and
permeabilized with Fix/Perm (Biolegend Cat #421002) according to
the manufacturer's instructions to stain for intracellular
IFN.gamma. (Clone B27 PE, Biolegend Cat#506507), and TNF.alpha.
(clone Mab11 APC, Biolegend Cat #502912). Cells were analyzed on a
Cytek Aurora spectral flow cytometer.
[0813] Ab-Dependent Cell Phagocytosis with Monocytes and
Neutrophils (ADCP and ADNP):
[0814] ADNP: HL-60 promyeloblast cells (ATCC Cat #CCL-240 were
maintained in Iscove's Modified Dulbecco's Medium (ATCC Cat
#30-2005) with 20% fetal bovine serum and 1% Pen/Strep. HL-60 cells
were differentiated into neutrophils by growth for 5 days in the
presence of 1.3% DMSO. Recombinant SARS-CoV-2 receptor binding
domain was coupled to fluorescent beads (Thermo Scientific Cat
#F8819) by carbodiimide coupling. Antibodies were diluted in a
five-fold dilution curve in HL-60 culture medium (1 .mu.g/ml-0.32
ng/ml) and incubated with RBD-coated beads for 2 h at 37.degree. C.
(5.times.10.sup.4 cells/well) were incubated for 18 h at 37.degree.
C. Cells were then stained for CD11b (Clone M1/70 APC-Fire750;
Biolegend Cat #101262) and CD16 (Clone 3G8 Pacific Blue, Biolegend
Cat#302032) and fixed with 4% paraformaldehyde, and analyzed by
flow cytometry. CD11b.sup.+ and CD16.sup.+ cells were analyzed for
uptake of fluorescent beads. A phagocytic score was determined
using the following formula: (percentage of
FITC*cells).times.(geometric mean fluorescent intensity (gMFI) of
the FITC+cells)/100,000.
[0815] ADCP: THP-1 monocytes were maintained in RPMI-1640
supplemented with 10% FBS, 1% Pen/Strep, 1% L-glutamine, and
b-mercaptoethanol. Recombinant SARS-CoV-2 RBD-coated beads were
generated as described for ADNP. Antibodies were diluted in a
five-fold dilution curve in THP-1 culture medium to (5 .mu.g/ml-64
.mu.g/ml) and incubated with RBD-coated beads for 2 h at 37.degree.
C. Unbound antibodies were removed by centrifugation prior to the
addition of THP-1 cells at 2.5.times.10.sup.4 cells/well. Cells
were fixed with 4% paraformaldehyde and analyzed by flow cytometry.
A phagocytic score was determined as described above.
[0816] Ab-Mediated Complement Deposition (ADCD):
[0817] Recombinant SARS-CoV-2 receptor binding domain-coated beads
were generated as described for ADNP. Antibodies were diluted in a
five-fold dilution series in RPMI-1640 (5 .mu.g/ml-64 .mu.g/ml) 5
.mu.g/ml and incubated with RBD-coated beads for 2 hours at
37.degree. C. Unbound antibodies were removed by centrifugation
prior to the addition of reconstituted guinea pig complement
(Cedarlane Labs Cat #CL4051) diluted in veronal buffer supplemented
with calcium and magnesium (Boston Bioproducts Cat #IBB-300) for 20
minutes at 37.degree. C. Beads were washed with PBS containing 15
mM EDTA, and stained with an FITC-conjugated anti-guinea pig C3
antibody (MP Biomedicals Cat #855385). C3 deposition onto beads was
analyzed by flow cytometry. The gMFI of FITC of all beads was
measured.
[0818] ADI-58124 and ADI-58125 were compared for the ability to
induce antibody dependent cellular cytotoxicity, as measured by
CD16a activation. As shown in FIG. 43F, which provides CD16a
activation upon binding to SARS-CoV-2 S protein (top) or SARS-CoV-2
S protein RBD (bottom), both ADI-58124 and ADI-58125 demonstrated
comparable ADCC activity. The results also indicate that CD16
activation is driven by engagement of the RBD.
[0819] Based on the results in FIGS. 43A-43E, the M428L/N434A
modification in the Fc region to extend half-life of ADI-58125 did
not alter Fc effector functions of the wild-type Fc-containing
ADI-58124 antibody. Moreover, ADI-58125 exhibits multiple Fc
effector activities, which may be critical for the clearance and
control of SARS-CoV-2 infection.
[0820] A brief description of the method used to assess ADCC are
provided below.
[0821] ADCC Activity Evaluation:
[0822] The ADCC assay assessed the activation of Jurkat-Lucia
effector cells (Invivogen, jktl-nfat-cd16) via antibody
cross-linking to antigen coated on high-binding 96 well plates
(Corning, catalog #3361). The Jurkat-Lucia cells have stable
expression of the cell surface Fc receptor CD16A (FcgRIIIA; V158
high affinity allotype). The Jurkat-Lucia cells also stably express
the Lucia luciferase reporter gene under the control of an ISG54
minimal promoter fused to six NFAT response elements. Jurkat-Lucia
binding to Fc of antibody-antigen complexes activates CD16A leading
to luciferase expression.
[0823] SARS-CoV-2 S protein (Hexapro S-2P as described in (Hsieh,
C. L., et al., (2020). Structure-based Design of
Prefusion-stabilized SARS-CoV-2 Spikes. bioRxiv.
doi:10.1101/2020.05.30.125484) or RBD (Wrapp, D. et al., (2020).
Science, 367(6483), 1260-1263. doi:10.1126/science.abb2507) was
coated on plates the day before beginning the assay. The day of the
assay, the coated plate was blocked and antibody dilutions (5
concentrations, 1:3 dilutions, from 2000 ng/mL to 25 ng/mL) were
added to the plate with controls. Next Jurkat-Lucia cells were
added at 1.times.105 cells/well, and plates were incubated at
37.degree. C. with 5% CO2. Samples were assayed 24 hours later for
luciferase activity by mixing assay supernatant with QUANTI-Luc
luciferase substrate (Invivogen, rep-qlc1). Luminescence was
measured using a SpectraMax Paradigm, Molecular Devices.
Example 30: In Vivo Effects by Post-Affinity Maturation
Antibodies
[0824] We tested the ability of ADI-58125 to provide broad in vivo
protection in an immunocompetent mouse model of COVID-19 using
mouse-adapted SARS-CoV (MA15) and mouse adapted SARS-CoV-2 (MA10)
(A. Roberts et al., PLoS Pathog 3, e5 (2007).; S. R. Leist et al.
Cell, (2020)). Balb/c mice were prophylactically treated with
either 200 .mu.g of ADI-58125 or PBS via IP injection 12 hours
prior to intranasal challenge with a 103 PFU dose of MA15 or MA10.
All mice were monitored daily for weight loss and changes in
respiratory function and groups of mice were euthanized at day two
or four post-infection to allow for measurement of virus
replication in the lung and analysis of lung histopathology.
Substantial, progressive weight loss in sham-treated mice infected
with both viruses along with increases in Penh, a calculated
measure of airway resistance (Leist S. R et al., A Mouse-Adapted
SARS-CoV-2 Induces Acute Lung Injury and Mortality in Standard
Laboratory Mice Mice. Cell (2020)) was observed. In contrast, mice
treated prophylactically with ADI-58125 demonstrated minimal weight
loss, no change in Penh and no signs of gross pathology at the time
of harvest (FIGS. 44A and 44B). Furthermore, prophylactic antibody
treatment prevented viral replication in the lungs at both two and
four days post infection (dpi). Next, the ability of ADI-58125 to
act anti-virally against SARS-CoV-2 MA10 in a therapeutic setting
was investigated. Mice were treated with 200 .mu.g of ADI-58125 or
PBS 12 hours following intranasal challenge with a 103 PFU dose of
MA10. Mice given therapeutic ADI-58125 had intermediate levels of
weight loss, moderate respiratory function changes and some gross
lung pathology; significantly more than prophylactically treated
mice but significantly less than sham-treated mice (FIG. 44C).
Therapeutic antibody treatment also resulted in a significant
reduction in lung viral loads at four dpi, but not at two dpi,
relative to sham-treated mice. In conclusion, ADI-58125 treatment
can reduce disease burden in mice infected with both SARS-CoV MA15
and SARS-CoV-2 MA10.
[0825] Neutralization of mouse adapted SARS-CoV and SARS-CoV-2 by
ADI-58125 was confirmed as shown in FIG. 44D, via a
luciferase-based assay using Vero E6 cells.
[0826] A brief description of the in vivo method used is provided
below.
[0827] Animal Studies:
[0828] Twelve-month old female Balb/c mice (Envigo, strain 047)
were treated with 200 .mu.g of ADI-58125 IgG via intraperitoneal
(IP) injection at either 12 hours prior to infection (prophylactic)
or 12 hours post-infection (therapeutic). Mice were anesthetized
with ketamine/xylazine before being challenged with 1000 PFU of
either SARS-CoV-MA15 or SARS2-CoV-2-MA10 (34, 35) via intranasal
inoculation. Mouse body weights and respiratory function were
monitored daily for 4 days. Respiratory function was monitored by
whole body plethysmography (DSI) with a 30-minute acclimation
period and a 5-minute measurement window as previously described
(51). Viral lung titer was measured by plaque assay, assessing the
lower lobe of the right lung. Gross pathology was performed on mice
sacrificed on day 2 and day 4 post-infection. Gross pathology in
the lung scored using a 4-point system, in which 0 represents no
hemorrhage and 4 represents complete and total hemorrhage. All
animal husbandry and experiments were performed at BSL3 and in
accordance with all University of North Carolina at Chapel Hill
Institutional Animal Care and Use Committee guidelines (AAALAC
Institutional Number 329).
[0829] Mouse Adapted Virus Neutralization:
ADI-58125 was diluted in growth media at 8 different concentration
to obtain a potential inhibition curve. SARS1-MA-nLuc (75 PFU/well)
and SARS2-MA2-nLuc (85 PFU/well) viruses were mixed with ADI-58125
at 1:1 and incubated. Virus and ADI-58125 mix were added to wells
of plated Vero E6 cells and incubated. Luciferase activities were
measured and percent inhibition, including IC50, was calculated by
fitting the data points using a sigmoidal dose response curve.
Example 31: Potential ADE Effects
[0830] Studies on SARS-CoV and other respiratory viruses poses a
question whether anti-SARS-CoV-2 antibodies could cause
antibody-dependent enhancement (ADE), in which antibodies are taken
up by immune cells via Fc receptors to result in increase virus
proliferation and/or enhanced inflammation. To test the possibility
of the antibodies according to the present disclosure and clinical
antibodies specific to SARS-CoV-2 to induce ADE, uptake of
pseudoviruses by phagocytes in the presence of the test antibodies
were examined. As shown in FIGS. 45A-C, ADI-58124 and ADI-58125 did
not mediate ADE of infection in both THP-1 and Raji cells.
[0831] The methods used in the ADE analyses are briefly provided
below.
[0832] Reporter Viral Particle Production:
[0833] CoV2pp, VSV .DELTA.G (rLuc) reporter virus particles, or
RVPs, expressing SARS-CoV-2 S were provided by Dr. Benhur Lee
(Icahn School of Medicine at Mount Sinai, New York City, N.Y.).
Production and titration of SARS-COV-2 RVPs is described in
(Oguntuyo K. Y. et al., Quantifying absolute neutralization titers
against SARS-CoV-2 by a standardized virus neutralization assay
allows for cross-cohort comparisons of COVID-19 sera. Version 2.
medRxiv. Preprint. 2020 Aug. 15 [revised 2020 Aug. 27]). Briefly,
293T cells were transfected to overexpress SARS-COV-2
glycoproteins. After suitable incubation, post transfected cells
were infected with the VSV-deltaG-rLuc reporter virus. Two days
post infection, supernatants were collected. VSVdeltaG-rLuc
particles bearing CoV2pp were processed and stored.
[0834] Antibody-Dependent Enhancement Assay:
[0835] Antibody-dependent enhancement assays were performed in Dr.
Paul Guyre's lab at the Geisel School of Medicine, Dartmouth
College, Lebanon, N.H. In vitro ADE was determined by assessing
uptake of the RVPs into THP-1 (human monocyte derived cell line,
ATCC TIB-202) or Raji cells (human B-cell derived cell line, ATCC
CCL-86) in the presence of a range of antibody concentrations from
above the IC50 to approximately 100-fold below the IC50. Antibody
dilutions were prepared in an opaque white 96-well plate (BRAND
plates, catalog number 781965) using serum-free, dye-free RPMI
media (Gibco, 11835-030) as diluent. A series of test antibody
concentrations starting at 100 ng/mL with 1:5 serial dilutions were
prepared with final antibody concentrations of 100, 20, 4, 0.8,
0.16, and 0.032 ng/mL. A control antibody was tested at the highest
concentration only (100 ng/mL).
[0836] The RVPs, CoV2pp, VSV .DELTA.G (rLuc), were thawed at
37.degree. C. for 3 min, then placed on ice prior to addition the
antibody plates. 20 .mu.L of RVP was added to each well. The RVPs
and antibody mixes were incubated at 37.degree. C., 5% CO2, for 1
hour. Then, Raji or THP-1 cells were added at a concentration of
2e5 cells/well, and plates were incubated for 18-24 hours at
37.degree. C., 5% CO2.
[0837] To measure luciferase activity associated with RVP uptake,
the Renilla Luciferase Assay kit was used (Promega, E2820). Lysis
buffer and renilla luciferase assay reagent were prepared from the
luciferase assay kit per manufacturer instructions. Plates were
removed from the incubator and centrifuged for 5 min at 1200 rpm to
pellet cells. Cells were washed with phosphate buffered saline
(PBS, Corning, 21-040-CV), then cells were pelleted by
centrifugation for 5 min at 1200 rpm and PBS was aspirated from all
wells. Cells were lysed using 50 .mu.L of 1.times.Lysis buffer, and
plates were incubated for 15 min on a rocker. A SpectraMax
i3.times. luminometer used to inject luciferase assay reagent (100
.mu.L) to each well, followed by immediate measurement of
luminescence. Data were plotted by histogram using GraphPad Prism
8.
Example 32: In Vivo Therapeutic and Prophylactic Efficacy of
Antibody Combinations
[0838] Therapeutic and/or Prophylactic Efficacy
[0839] Test animals, preferably mammals such as mice, rats, hamster
(e.g., Syrian hamsters), rabbits, pigs, or monkeys, will be split
in multiple groups. ADI-57983, ADI-57978, ADI-56868, ADI-56443, and
ADI-56479 or fragment thereof or a mixture of such IgGs and/or
fragments thereof, especially antibodies that recognize different
epitopes and/or do not compete with each other, e.g., "ADI-57983
and ADI-56443", will be administered to at least one group. At
least one group will not receive such antibody or fragment thereof.
The animals will then be infected with coronavirus (e.g., SARS-CoV,
SARS-CoV-2 etc). Alternatively, the antibody or fragment thereof
may be administered before the infection with CoV. The antibody or
fragment thereof may be given intravenously, intraperitoneally,
intranasally, subcutaneously, or via any other appropriate
route.
[0840] The body weight of each animal will be monitored. Symptoms
such as fever or mobility may also be monitored. Periodically,
samples such as serum will be harvested and the viral load will be
measured. Survival will be tracked. Animals may be sacrificed based
on the pre-determined cutoff value of the body weight and/or
viremia and/or the behavior and/or symptom(s).
[0841] Grouping that may be used for in vivo studies include, but
is not limited to:
[0842] Group 1, negative control;
[0843] Group 2, ADI-58124 (or its Fc variant) only, ADI-58124 (or
its Fc variant) only, or ADI-58126 (or its Fc variant) only
(RBD-binding broad neutralizer);
[0844] Group 3, ADI-581230 (or its Fc variant) only (NTD
binder);
[0845] Group 4, ADI-58124 (or its Fc variant)+ADI-581230 (or its Fc
variant) (RBD-binding broad neutralizer+NTD binder);
[0846] Group 5, ADI-58128 (or its Fc variant) only (RBD binder, no
competition with ADI-57983);
[0847] Group 6, ADI-58124 (or its Fc variant)+ADI-58128 (or its Fc
variant) (RBD-binding broad neutralizer+non-competing RBD
binder);
[0848] Group 7, ADI-58124 (or its Fc variant)+ADI-581230 (or its Fc
variant) (NTD binder)+ADI-58128 (or its Fc variant) (RBD binder, no
competition with ADI-57983).
[0849] Matching Fc will be used between Groups to allow for proper
comparison.
[0850] A therapeutic or prophylactic dose of below 1 mg, for
example 3 .mu.g, may be administered.
Example 33: A Phase 2/3 Randomized, Double-Blind,
Placebo-Controlled Clinical Trial to Evaluate the Efficacy and
Safety of ADI-58125 in the Prevention of COVID-19
[0851] A phase 2/3 randomized, double-blind, placebo-controlled
trial is designed to evaluate the efficacy and safety of ADI-58125
in the prevention of RT-PCR confirmed, symptomatic COVID-19.
[0852] The study involves 5,716 adult and adolescent participants
whose locations or circumstances place them at high risk of
acquiring SARS-CoV-2 and COVID-19. Baseline serology/RT-PCR will be
collected with regards to SARS-CoV-2 status. The duration of this
study is about 12 months for each participant which corresponds to
12 months follow-up after receipt of the treatment (300 mg
ADI-58125 or placebo). The treatment (ADI-58125 or placebo) is
given intramuscularly. [0853] Treatment groups are divided as
following:
TABLE-US-00005 [0853] Dosage IM Strata (once) 1:1 Sample size Known
recent exposure to confirmed 300 mg, placebo 40% case No known
recent exposure 300 mg, placebo 60% .gtoreq.65 years 300 mg,
placebo 12 to <65 years at increased risk for 300 mg, placebo
severe COVID-19 ("at risk") 12 to <65 and not at risk 300 mg,
placebo
[0854] The results of this study demonstrate the immediate and
durable efficacy of ADI-58125 in preventing COVID-19 in a broad
population.
Example 34: A Phase 1/2/3 Randomized, Double-Blind,
Placebo-Controlled Clinical Trial to Evaluate the Efficacy and
Safety of ADI-58125 in the Treatment of Ambulatory Participants
with Mild or Moderate COVID-19
[0855] A phase 1/2/3 randomized, double-blind, placebo-controlled
trial is designed to evaluate the efficacy and safety of ADI-58125
in the treatment of mild or moderate COVID-19 in participants at
high risk of disease progression.
[0856] This study involves 1,734 high risk adult and adolescent
participants with mild or moderate COVID-19 with symptom duration
of 5 days or less and a positive SARS-CoV-2 test. The study lasts
for approximately 6 months for each participant which corresponds
to 6 months follow-up after receipt of the treatment (ADI-58125 or
placebo). The treatment (ADI-58125 or placebo) is given either
intravenously or intramuscularly.
[0857] Treatment groups are divided as following:
TABLE-US-00006 Strata Dosage (once) 2:2:1:1 >65 years 600 mg IM,
1200 mg IV, placebo IM, placebo IV 18 to 65 years 300 mg, placebo
12 to 17 300 mg, placebo Geographic 600 mg IM, 1200 mg IV, location
placebo IM, placebo IV
Example 35: Dose Regimen Selection by a Model Based Approach
[0858] A QSP/PBPK model was developed and modified to characterize
extended half-life monoclonal antibody pharmacokinectics (PK),
including the impact of drug-specific physiochemical properties.
Non-human primate PK data was used to evaluate the model through
comparison of predicted and measured concentrations. The model was
then re-fitted to better describe the measured PK and used to
select doses for the prevention trial in human.
[0859] To support the dose justification for treatment, the
QSP/PBSK model was further modified by replacing the lung
compartment with distinct upper and lower airway compartments, the
two key sites of ADI-58125 interaction with SARS-Cov-2, and linking
the model to viral time-course of infection model.
[0860] For dose selection for prevention, greater than 90% of
simulated patients were predicted to maintain serum ADI-58125
concentrations above 100 to 200 times of the in vitro IC90 for a
minimum of 6 months following a single 300 mg intramuscular (IM)
dose (FIG. 46). Doses of 150 and 450 mg showed similar results
(data not shown). Similarly, greater than 90% of simulated patients
were expected to maintain ADI-58125 lung interstitial fluid
concentrations greater than 18 times of the in vitro IC90 for over
6 months. Greater than 90% of simulated patients were expected to
maintain ADI-58125 concentrations greater than 500 times of the in
vitro IC50 for a minimum of 6 months. This threshold is similar to
the 50% SARS-CoV-2 geometric mean titer observed on Day 35 (7 days
after second vaccine dose) with the COVID-19 vaccine BNT62b1
(Mulligan, 2020), which was recently announced to have achieved
>90% efficacy in the prevention of COVID-19 at 7 days after the
second dose (Pfizer, 2020).
[0861] For dose selection for treatment, greater than 90% of
simulated patients were predicted to maintain serum neutralizing
antibody concentrations greater than 500 times of the in vitro IC50
and upper and lower respiratory ELF concentrations greater than 100
times of the in vitro IC.sub.50 for a minimum of 28 days following
a single dose of 600 mg IM (FIG. 47A) or a single dose of 1200 mg
IV over 1 hour (FIG. 47B). In contrast, the LY-CoV555 regimen
maintains serum neutralizing antibody concentrations greater than
500 times of the in vitro IC50 for only 21 days in >90% of
patients (FIG. 47C) and neither the LY-CoV555 or REGN10987 regimen
maintains ELF targets beyond Day 21 in >90% of patients (FIG.
47D).
[0862] The impact of the selected treatment dosage for ADI-58125
(600 IM and 1200 mg IV) on viral dynamics was also assessed
relative to that of REGN-COV2 (casirivimab/imdevimab) 2400 mg IV in
subjects with a high baseline viral load (>107 copies/mL). The
median effect of the ADI-58125 600 mg IM dose approached that of
the REGN-COV2 regimen over a 1-day period (FIG. 48A); while the
ADI-58125 1200 mg IV dose matched the effect of the REGN-COV2
regimen (FIG. 48B). The probability of ADI-58125 600 mg IM and
ADI-58125 1200 mg IV matching the viral load change observed with
the REGN-COV2 regimen was simulated in 1000-patients for each
proposed dose level. The ADI-58125 600 mg IM dose approached 90% of
the effect of the REGN-COV2 regimen over a 2-day period (FIG. 48C)
and the ADI-58125 1200 mg IV dose matched the effect of the
REGN-COV2 regimen immediately (FIG. 48D). While it is unknown if
the delay in reaching maximal effect for the 600 mg IM dose impacts
clinical outcomes, it is important to note that approximately 66%
and 90% of this regimen's maximal effect was predicted to be
achieved by 12 and 24 hours, respectively. Study of the IM regimen
is further supported by the potential benefits to patients,
providers and healthcare systems alike given the relative ease of
administration of this regimen in the outpatient setting for
ambulatory patients with COVID-19.
Example 36: A Phase 1, Randomized, Double-Blind, Single Ascending
Dose Study Evaluating Safety, Tolerability, and PK of ADI-58125 in
Healthy Participants
[0863] A phase I, randomized, double-blinded, single ascending dose
study is designed to evaluate the safety, tolerability and
pharmacokinetics of ADI-58125 in healthy participants.
[0864] This study involves healthy volunteers with an age of about
18-50. Participants are divided into three dose cohorts: 300 mg IM
(intramuscular), 500 mg IV (intravenous), or 600 mg IM. Each cohort
comprises 10 participants, where 8 individuals receive active
treatment and 2 individuals receive a placebo control.
Example 37: A Non-Clinical Safety Study
[0865] In a GLP rat 22-day repeat-dose study, there were no
ADI-58125-associated toxicity findings, including no local
injection site reactions. No off-target binding of ADI-58125 to
human tissues was observed in a human tissue cross-reactivity IHC
study, supporting use of the rat as an appropriate species for
toxicity assessment.
Human Cross-Tissue Reactivity Data
[0866] Immunohistochemistry (IHC) staining methods were used to
determine the binding activity of the biotinylated test article,
Biotin-ADI-58125, and control article, Biotin-562, with a panel of
37 different frozen normal human tissues. Phosphate-buffered saline
(PBS, 0.1 mol/L) was the reagent control. Streptavidin-peroxidase
and stable diaminobenzidine (DAB) were used for color development.
Positive control cells were 200221_165_S9sh) cells (SARS-CoV-2
Spike protein transfected HEK293 cells) and negative cells were
non-transfected HEK293 cells. Both test article and control article
were biotinylated during method development and validation study.
Frozen normal human tissues were validated with anti-CD31
(anti-endothelial cells) monoclonal antibody staining.
[0867] No specific Biotin--ADI-58125 staining was observed in
frozen human tissues and no human tissue sections had positive
membrane staining by IHC with Biotin--ADI-58125. Therefore, there
was no cross-reactivity (off-target binding) of ADI-58125 to normal
human tissues.
GLP Rat Toxicity Study
[0868] One hundred and seventy Sprague-Dawley (SD) rats (85/sex)
were randomly assigned to 4 groups to determine the toxicity and
toxicokinetics of ADI-58125 when administered once weekly (on Days
1, 8, 15, and 22) by IV infusion (30 or 300 mg/kg) or IM injection
(30 mg/kg) compared to control group. Five rats/sex from 300 mg/kg
IV, 30 mg/kg IM, and control group were allocated for a 21-day
recovery observation. The scheduled necropsied were conducted on
Days 23 (dosing phase) and 44 (recovery phase). Criteria for
evaluation included viability (morbidity and mortality), clinical
observations, body weight, food consumption, clinical pathology
(hematology, serum chemistry, coagulation, and urinalysis), organ
weight, gross observations, histopathology evaluation, and TK.
Study design was agreed to with the FDA prior to initiation.
[0869] Once-weekly administration of ADI-58125 to adult rats for 22
days (4 doses in total) by IV infusion up to 300 mg/kg/dose or by
IM injection at 30 mg/kg/dose did not result in any test article
related mortality or adverse effects. The no observed adverse
effect level (NOAEL) was considered to be 300 mg/kg/dose for IV
infusion and 30 mg/kg/dose for IM injection.
Example 38: A Preclinical PK/PD Study: Non-GLP Pharmacokinetics in
Non Human Primates
[0870] A non-GLP pharmacokinestic study was performed in non human
primates (NHPs). As shown in FIG. 49A, there was no sex differences
in PK or mean serum concentration after IV infusion or IM injection
of ADI-58125 at a dose of 10 mg/kg in male and female cynomolgus
monkeys.
[0871] Pharmacokinetic parameters were evaluated. A long half-life
was confirmed with an average of about 473 h following IV
administration and about 533 h following IM administration, as well
as a bioavailability of .about.100% (FIG. 49B).
Example 39: ADI-58122 and ADI-58125 Potently Neutralize UK
(B.1.1.7), South African (B.1.351) Brazilian (B.1.1.128), and
B.1.429 (Southern California) SARS-CoV-2 Variants
[0872] Neutralization assays were performed for ADI-58122 and
ADI-58125 on the UK (B.1.1.7) and South African (B.1.351)
SARS-CoV-2 variants, as described above. As shown in FIGS. 50A-50C,
both ADI-58122 and ADI-58125 potently neutralize UK (B.1.1.7),
South African (B.1.351) and/or Brazilian (P.1) SARS-CoV-2 variants
with an IC50 less than 0.05 .mu.g/mL and with 100% neutralization
plateau. These two broad neutralizers had a lower neutralization
IC50 value against the UK and South African strains than most of
the SARS-CoV-2 only neutralizers (FIG. 51).
[0873] Binding affinity was also assessed for ADI-58125 against the
Brazilian SARS-CoV-2 variant (B.1.1.128) and the newly emerging
variant in southern California (B.1.429) in addition to the UK
(B.1.1.7) and South African (B.1.351) SARS-CoV-2 variants. As shown
in FIGS. 52A and 52B, ADI-58125 retains high binding affinity to
the RBDs of all four varaints, whereas other antibodies, for
example, LYCoV-555, LYCoV-016 and REGN10933, were less effective in
binding to the RBD of the variants, for example the South African
variant or the southern California variant.
Example 40: In Vivo Efficacy Study in Hamster
[0874] The prophylactic efficacy of ADI-58125 was assessed in vivo
in hamster. Briefly, 5-6 week old female Syrian hamsters were dosed
intraperitoneally with a range of ADI-58125 doses (n=40; 9.25-2000
.mu.g) or control mAb (sham isotype matched IgG) (n=20; either 9.25
or 2000 .mu.g) 24 hours prior to intranasal challenge with 1e5 pfu
of SARS-2/WA-1 to evaluate prophylactic efficacy of ADI-58125 (FIG.
53A). Prior to viral challenge, serum antibody titres were
measured. The prophylactic efficacy of ADI-58125 was assessed by
viral load (plaque assay, genomic RT-PCR, subgenomic RT-PCR), body
weight, and histopathology. Hamsters were weighed daily over 6
days. On days 3 and 6, antibody titre, viral load and lung
histopathology were assessed.
[0875] As shown in FIG. 53B, an ADI-58125 dose dependent decrease
in viral load was observed. Specifically, hamsters receiving the
highest dose (2000 .mu.g) had no detectable virus in lung samples.
Similar trends were observed for genomic-RNA and sub-genomic-RNA
(data not shown). An ADI-58125 dose of .gtoreq.55 .mu.g was
associated with protection from weight loss compared with controls
(FIG. 53C), and hamsters receiving 333 and 2000 .mu.g doses
displayed limited histopathological evidence of pneumonia (data not
shown). These data demonstrated that prophylactic administration of
ADI-58125 provides dose-dependent protection from SARS-CoV-2
infection in the Hamster model.
Example 41: In Vivo Efficacy Study in Non Human Primates
[0876] The prophylactic efficacy of ADI-58125 was further assessed
in vivo in non human primates (NHP). Briefly, rhesus macaques
(>3 years of age at time of challenge; 3-10 kg in weight; 4/arm)
were dosed intravenously with ADI-58125 at 5 mg/kg or 25 mg/kg
(n=8), or control mAb (25 mg/kg, n=4) 3 days prior to
intranasal/intratracheal challenge with 1e6 pfu of SARS-2/WA-1 to
evaluate prophylactic efficacy of ADI-58125 (FIG. 54A). Prior to
viral challenge, blood samples were collected and daily
pharmacokinetic samples and viral (nasopharyngeal, oropharyngeal
(daily) and broncholaveolar lavage (days 1, 3, and 5) were
assessed. The prophylactic efficacy of ADI-58125 was assessed by
viral load (plaque assay, genomic RT-PCR, subgenomic RT-PCR),
clinical disease on chest radiograph and histopathology. In the NHP
model, accelerated clearance of genomic RNA with no detection of
sub-genomic RNA was observed at the 20 mg/kg dose in both
nasopharyngeal and bronchoalveolar lavage samples, demonstrating a
significant impact of ADI-58125 on viral replication in the upper
and lower airways (FIG. 54B). No evidence of enhanced viral
replication at any ADI-58125 dose level was observed in either
hamster or NHP animal model. These data demonstrated that ADI-58125
confers potent protection from SARS-CoV-2 infection at dose ranges
from 5-25 mg/kg in a NHP model. These results support further
investigation of ADI-58125 for the prevention of COVID-19 in
humans.
TABLE-US-00007 TABLE 5 Summary of Antibody names (ADI ID) and Index
Nos. Index No. ADI ID 1 ADI-55688 2 ADI-55689 3 ADI-55690 4
ADI-55691 5 ADI-55692 6 ADI-55693 7 ADI-55694 8 ADI-55695 9
ADI-55696 10 ADI-55697 11 ADI-55698 12 ADI-55699 13 ADI-55700 14
ADI-55701 15 ADI-55702 16 ADI-55703 17 ADI-55704 18 ADI-55705 19
ADI-55706 20 ADI-55707 21 ADI-55708 22 ADI-55709 23 ADI-55710 24
ADI-55711 25 ADI-55712 26 ADI-55713 27 ADI-55714 28 ADI-55715 29
ADI-55716 30 ADI-55717 31 ADI-55718 32 ADI-55719 33 ADI-55721 34
ADI-55722 35 ADI-55723 36 ADI-55724 37 ADI-55725 38 ADI-55726 39
ADI-55727 40 ADI-55728 41 ADI-55729 42 ADI-55730 43 ADI-55731 44
ADI-55732 45 ADI-55733 46 ADI-55734 47 ADI-55735 48 ADI-55736 49
ADI-55737 50 ADI-55738 51 ADI-55739 52 ADI-55740 53 ADI-55741 54
ADI-55742 55 ADI-55743 56 ADI-55744 57 ADI-55745 58 ADI-55746 59
ADI-55747 60 ADI-55748 61 ADI-55749 62 ADI-55750 63 ADI-55751 64
ADI-55752 65 ADI-55753 66 ADI-55754 67 ADI-55755 68 ADI-55756 69
ADI-55757 70 ADI-55758 71 ADI-55720 72 ADI-55760 73 ADI-55761 74
ADI-55762 75 ADI-55763 76 ADI-55765 77 ADI-55766 78 ADI-55767 79
ADI-55769 80 ADI-55770 81 ADI-55771 82 ADI-55775 83 ADI-55776 84
ADI-55777 85 ADI-55950 86 ADI-55951 87 ADI-55952 88 ADI-55953 89
ADI-55954 90 ADI-55955 91 ADI-55956 92 ADI-55957 93 ADI-55958 94
ADI-55959 95 ADI-55960 96 ADI-55961 97 ADI-55962 98 ADI-55963 99
ADI-55964 100 ADI-55965 101 ADI-55966 102 ADI-55967 103 ADI-55968
104 ADI-55969 105 ADI-55970 106 ADI-55972 107 ADI-55973 108
ADI-55974 109 ADI-55975 110 ADI-55976 111 ADI-55977 112 ADI-55978
113 ADI-55979 114 ADI-55980 115 ADI-55981 116 ADI-55982 117
ADI-55984 118 ADI-55986 119 ADI-55988 120 ADI-55989 121 ADI-55990
122 ADI-55992 123 ADI-55993 124 ADI-55994 125 ADI-55995 126
ADI-55996 127 ADI-55997 128 ADI-55998 129 ADI-55999 130 ADI-56000
131 ADI-56001 132 ADI-56002 133 ADI-56003 134 ADI-56004 135
ADI-56005 136 ADI-56006 137 ADI-56007 138 ADI-56008 139 ADI-56009
140 ADI-56010 141 ADI-56011 142 ADI-56012 143 ADI-56013 144
ADI-56014 145 ADI-56015 146 ADI-56016 147 AD1-56017 148 ADI-56018
149 ADI-56019 150 ADI-56020 151 ADI-56021 152 ADI-56022 153
ADI-56023 154 ADI-56024 155 ADI-56025 156 ADI-56026 157 ADI-56027
158 ADI-56028 159 ADI-56029 160 ADI-56030 161 ADI-56031 162
ADI-56032 163 ADI-56033 164 ADI-56034 165 ADI-56035 166 ADI-56037
167 ADI-56038 168 ADI-56039 169 ADI-56040 170 ADI-56041 171
ADI-56042 172 ADI-56043 173 ADI-56044 174 ADI-56045 175 ADI-56046
176 ADI-56047 177 ADI-56048 178 ADI-56049 179 ADI-56050 180
ADI-56051 181 ADI-56052 182 ADI-56053 183 ADI-56054 184 ADI-56055
185 ADI-56056 186 ADI-56057 187 ADI-56058 188 ADI-56059 189
ADI-56061 190 ADI-56062 191 ADI-56063 192 ADI-56064 193 ADI-56065
194 ADI-56066 195 ADI-56067 196 ADI-56068 197 ADI-56069 198
ADI-56070 199 ADI-56071 200 ADI-56072 201 ADI-56073 202 ADI-56074
203 ADI-56075 204 ADI-56076 205 ADI-56078 206 ADI-56079 207
ADI-56080 208 ADI-56081 209 ADI-56082 210 ADI-56083 211 ADI-56084
212 ADI-57983 (with primer mutation) 213 ADI-57978 (with primer
mutation) 214 ADI-56868 (with primer mutation) 215 ADI-56443 (with
primer mutation) 216 ADI-56479 (with primer mutation) 217 ADI-57983
(Fc variant: WT) 218 ADI-57983 (Fc Variant: YTE) 219 ADI-57983 (Fc
Variant: LA) 220 ADI-57983 (Fc Variant: LS) 221 ADI-57983 (Fc
Variant: LA-RE) 222 ADI-57978 (Fc Variant: WT) 223 ADI-57978 (Fc
Variant: LA) 224 ADI-56868 (Fc Variant: WT) 225 ADI-56868 (Fc
Variant: LA) 226 ADI-56443 (Fc Variant: WT) 227 ADI-56479 (Fc
Variant: WT)
[0877] Having fully described and enabled the invention, the
invention is further described by the claims that follow. In
general, in the following claims, the terms used should not be
construed to limit the disclosure to the specific embodiments
disclosed in the specification and the claims. Accordingly, the
invention is not limited by the disclosure, but instead the scope
of the invention is to be determined entirely by the following
claims.
[0878] The disclosure may be practiced in ways other than those
particularly described in the foregoing description and examples.
Numerous modifications and variations of the disclosure are
possible in light of the above teachings and, therefore, are within
the scope of the appended claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210324048A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210324048A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References